JPH0716419B2 - Rennin, pre-prorennin, or prorennin genes obtained from recombinant DNA material, and live cells containing these genes - Google Patents

Abstract

Living cells containing genetic material derived from recombinant DNA material and capable of expressing rennin, pre-prorennin and prorennin. The rennin, pre- prorennin and prorennin are derived from cells which are themselves or have had parents thereof treated by recombinant DNA methods to allow production of the desired anzymatic proteins during growth in culture.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to live cells containing genetic material derived from recombinant DNA material and capable of expressing rennin, pre-prorennin and prorennin. Rennin, pre-prorennin and prorennin are obtained either from the cells themselves or from their parental cells which have been treated by recombinant DNA methods to produce the desired enzyme protein during growth in culture.

The enzyme protein rennin has long been known to be useful in coagulating milk casein in cheese making.
Rennin is also used in connection with cheese ripening because of its unique proteolytic activity. In the past, rennin has been obtained from rennet for commercial production. Calves bred in milk are sacrificed and the food-free abomasum is removed. Then dry their stomachs, salt them,
It uses a complicated method of freezing. At the factory stage, the stomachs are cleaned, treated to separate salts and remove surface fat. The stomachs are then spread over a rack and dried. The dried stomachs are often stored refrigerated, then ground and placed in a large tank of brine solution circulating through the skin until rennin extraction is complete. The above process for producing rennin is expensive and involves many difficulties in producing the vast quantities required for commercial use in various applications throughout the world.

The object of the present invention is to obtain viable cells capable of producing rennin in culture for mass production.

Another object of the present invention is to obtain live cells capable of producing prorennin in culture for mass production.

Another object of the invention is to obtain viable cells capable of producing pre-prorennin in culture for mass production.

Yet another object of the present invention is to obtain a rennin, prorennin, or pre-form obtained from a living cell which is consistent with the above object, wherein the living cell contains genetic material obtained from recombinant DNA material.
Providing prorennin.

Still another object of the present invention is to provide a rennin gene, a pre-prorennin gene and a prorennin gene having a special function.

Yet another object of the present invention is to provide a method for producing rennin, prorennin or pre-prorennin using recombinant DNA technology.

Yet another object of the present invention is the use of a selected amino acid sequence, such as a selected enzyme or protein material, for extracellular transfer with a periplasmic space, other cellular compartments, or a compatible host. To provide a cryptographic array for.

Yet another object of the present invention is to provide special modified cells for use in the production of rennin or a polypeptide exhibiting milk coagulation activity.

According to the present invention, living cells contain genetic material derived from recombinant DNA material and are capable of expressing rennin, pre-prorennin, or prorennin. The invention also comprises rennin, prorennin and pre-prorennin obtained from living cells, and genes therefor.

According to the method of the present invention, expression of pre-prorennin in a host cell is obtained by generating a DNA sequence encoding pre-prorennin. The sequence binds to the transcriptional promoter and the ribosome binding site at its 5'end and is responsible for the origin of the DNA encoding pre-prorennin and the promoter and binding site.
The distance to the NA fragment changes. The DNA is then transferred (transformed) into a host cell. Host cells are cloned and host cells with high levels of expression of pre-prorennin selected.

In the method of obtaining prorennin or rennin expression in a host cell, a DNA sequence encoding pre-prorennin and having a 5'end is selected. 5'part
Removed from the end, the portion encodes a prorennin or rennin precursor polypeptide. The remaining portion with the prorennin or rennin coding sequence is ligated onto a synthetic fragment of DNA which has a translation initiation codon at the 3'end of the fragment. Then, in the same manner as described above, the operation of binding the transcription promoter and the ribosome binding site to the DNA sequence, and the initiation point of the DNA encoding prorennin or rennin and the fragment of DNA having the promoter and the ribosome binding site Continue the operation to change the distance between. This material is introduced into a host cell, cloned and then selected for cells expressing the desired and selected prorennin or rennin.

Escherichia coli (E. coli) prepared by the method described herein was Maryland 20852, Rotkville, 123.
It is exemplified by the deposited culture of the American Type Culture Collection at 01 Park Warren Drive and is identified as Accession No. 31929, a CGE24 strain derived from E. coli BNN45 strain.

Yeast microorganisms prepared by the methods described herein are exemplified by cultures deposited at the American Type Culture Collection of 12301 Park Warren Drive, Maryland 20852, Rottukville, 1230, and Saccharomyces It is identified as Accession No. 20623, which is a CGY116 strain derived from the S. cerevisiae CGY80 strain.

Preferably, in the method of the present invention, pre-prorennin, prorennin and rennin can each be obtained by isolation of pre-prorennin DNA material. Pre-prorennin is a precursor of prorennin and has not been described in the literature. By removing a portion of the pre-prorennin DNA, the genetic material encoding prorennin or rennin can be obtained.

The pre-prorennin, prorennin or rennin gene according to the invention contains a specific nucleotide sequence encoding the amino acid sequence of each of pre-prorennin, prorennin or rennin, and encodes each of pre-prorennin, prorennin or rennin Excludes all intervening nucleotide sequences present in genomic DNA. These three genes are also provided linked to a vector that replicates in a suitable host cell.

The host cell is preferably E. coli, which is capable of expressing recombinant DNA material that produces the desired enzyme protein. Yeast and other cells can also be used.
These cells are selected such that they can produce large quantities of the enzyme protein under reasonable commercial scale culture conditions.

The enzyme rennin described in this specification (EC3.4.23.4)
Is also known as chymosin. It is the major hydrolyzable protein found in the stomach of previously ruminant calves and is reactive to coagulated milk. Rennin is used commercially in the manufacture of cheese. Prorennin was reported by B. Foltmann et al. In Proc. Nat. Acad. Sci. USA.
It is a precursor form of rennin with 42 additional amino acids at the amino terminus as described in Vol. 74, pp. 2321-2324 (1977). The first described pre-prorennin in this specification is the precursor form of prorennin and has many additional amino acids (16 amino acids) on the amino terminus of the prorennin molecule. These additional amino acids are preferably important for the selection of enzymes from gastric cells. B. Foltmann
Et al. Showed that purified rennin is a mixture of two forms, A and B [B. Foltmann et al., Proc. Nat. Acad.S.
ci.USA Vol.74, pp.2321-2324 (1977) and J.Biol.Che.
m. 254, 8447-8456 [1979]. Both forms are active, and sequence data indicate that perhaps the only difference is the aspartate residue at position # 290 in rennin A and the glycine residue at that position in rennin B. . The rennin produced in the examples of the present invention is rennin A. However, rennin B can be produced by the same procedure and / or simple conversion. Similarly, the pre and pro forms are A or B
Can occur in shape.

For the purposes of the present invention, the prorennin gene is defined as the specific nucleotide sequence encoding the prorennin molecule, the amino acid sequence of which has been described in the literature [B. Foltmann. VB Pedersen, H. Jacobsen, D. Kaufman. , And G. Wybrandt, Proc. Nat. Acad. Sci. USA Vol. 74, 2321 ~ 2
324 (1977)].

The pre-prorennin gene not only has the sequence of nucleotides that encode prorennin, but also contains 48 additional nucleotides on the 5'end that encode the amino-terminal precursor polypeptide found on the pre-prorennin enzyme. To do.

The rennin gene is defined as the specific sequence of nucleotides that encodes the rennin molecule, which excludes the first 126 nucleotides that encode the proenninic portion of prorennin.

Live cells are produced in the first example by using multiple and known DNA techniques starting with material obtained from the abomasum of calves.

The resulting rennin, pre-prorennin and prorennin can be used to coagulate milk in cheese making to obtain cheese and possibly in other commercial applications to coagulate milk to obtain cheese What is possible is one of the features of the present invention. Mass production is possible by culture technology. In this way, large quantities of material can be produced at reasonable production rates and costs. The genetically modified DNA material is substantially identical to the rennin portion of the calf pre-prorennin gene when rennin is to be produced and when pre-prorennin is to be produced. Is substantially identical to the calf pre-prorennin gene and, when prorennin is to be produced, is substantially identical to the prorennin portion of the calf pre-prorennin gene. Differences in recombinant DNA are primarily associated with molecules that do not have the intron present in calf genes.

Bacteria of all species considered safe for recombinant DNA manipulation can be used. These bacteria include, for example, Escherichia coli (Escherichia coli), Bacillus of various species, such as Bacillus. Subtilis, various Lactobacillus species, and various Micrococcus species,
For example, Micrococcus fragilis is included. Other cells such as fungi, yeast or mammals can also be used as host cells. In each case, the resulting genetic information of the cells is the recombinant DN
Contains new genetic material derived from A material. This material is often included in the form of a plasmid, which is capable of replicating in the host cell and into which the genetic material from the donor cell is inserted at an early stage in the host cell. When a recombinant DNA molecule is formed,
Once inserted into a host cell, the host cell grows and replicates by essentially normal means. Production of an enzymatic protein (which may be rennin, pre-prorennin or prorennin) encoding recombinant DNA material occurs according to the present invention.

The host cell into which the desired recombinant DNA material is introduced is preferably an E. coli K-12-derived strain. The useful origin strains are as follows.

HB101, HWBoyer & D. Roulland-Dussoix (1969) J. Mol.
Biol. 41, 459-472, C600, M. Mandel & A. Higa J. Mo.
L. Biol. 53, 159-162 (1970), and strains derived from C600, such as MV1 and MV12, V. Hershfield, HWBoyer, C. Yanofsky, M.
A. Lovett & D.R. Helinski (1974) Proc.Natl.Acad.Sci.U
SA Vol. 71, pp. 3455-3459, LE392, SM Gilghman, DC Tiemeier, F. Polsky, MH Edgel
l, JGSeidman, A.Leder, LWEnquist, B.Norman & P.Lede
r (1977) Proc.Natl.Acad.Sci.USA Vol.74, 4406-4410
Page, JM101, J.Messing (1979) Recombinant DNA Technical B
ulletin Vol. 2, pp. 43-48, W3110 and derived strains, KL Korr & C. Yarofsky (1976) J. Mo.
l. Biol. 103, 395-409. These cells can be grown by conventional culturing techniques. For example, the medium for E. coli is as follows.

Nutrient medium Bacto tryptone per LB (Difco Laboratories, Detroit) 10g Bacto yeast extract (Difco Laboratories, Detroit) 5g NaCl 10g Glucose 2g TY per Bactto tryptone (Difco Laboratories, Detroit) 10g Bacto yeast (Difco Laboratories, Detroit) Detroit) 1g NaCl 8 g glucose 1g minimal medium M9 per _{NaHPO 4 6g KH 2 PO 4 3g} NaCl 0.5g CaCl 2 11mg MgCO 4 7H 2 O 0.2g Bl ( thiamine HCl) amino acids required amount by 5mg strain (each 40 mg) of glucose 2g Cultures can be grown in suspension and plated on agar, or other standard techniques and cell culture techniques can be used.

The medium used can be any standard medium for growing the particular cells. For example, E. coli LE392 (E. coli _{^{C600 ▲ r - k ▼ m +}} SupE, SupF, gal -) or BNN45 (E. coli ^{hadR -, hadM +, SupE,} SupF, Bl - met -) (Advanced Bact
erial Genetics, RWDavis, D, Botstein, JRRoth, Cold Spring Harbor Laboratory (1980) p.
TX medium initially seeded with cells of 7) at a level of 1% to 4% is preferred.

In standard techniques, the E. coli is 10-30 × 10 ¹² cells 1
The desired enzyme protein is produced by growing to the density of.

Various media known for growing cells can be used, but do not form part of the present invention. Similarly, various growth methods, techniques and conditions can be used. For example, the cells are preferably 30 ° C to 40 ° C
Temperatures outside this range can be used while growing at temperatures of.

The starting point for obtaining the cells of the present invention is recombinant DNA known in the art for obtaining the desired genetic material and for inserting it into a host cell and subsequently cloning the host cell.
Is to use technology.

Preferably, the rennin gene, the pre-prorennin gene or the prorennin gene, which is ultimately desired to be cloned in a microorganism, is obtained by obtaining a messenger RNA of the pre-prorennin gene from a tissue source.
Isolated in stages. In the case of calves, this is obtained by isolation from the abomasum of calves. Messenger RNA is Deele
can be isolated by the method of y et al. (RGDeeley,
JIGordon, ATHBurns, KPMullinix, M.Bina-Stein,
RF Goldberger J. Biol. Chem. 252, 8310-8319 (1
977]). Also, poly A-enriched RNA is described in RC Desrasiers, K .;
Oligo (dT) by the method of H. Friderici, & F.M. Rottman
It can be obtained by chromatography on cellulose (Biochemistry, Vol. 14, 4367-4374 [197].
Five〕).

The mRNA is then double-stranded by conventional means.
Converted to NA. First, the complement of the RNA (complimen
tary) copies are made from the mRNA by conventional recombinant DNA means, such as by using AMV reverse transcriptase. For example, A. Efstratiadis, FC Kafatos, AM Maxam and T. Maniatis, Cell, 7: 279-288 (1976), R.Hi.
guchi, GVPaddock, R. Wall and W. Salser, Proc. Nat. Ac
ad, Sci. USA Vol. 73, 3146-3150 (1976), DL Kacian and JC Myers, Proc. Nat. Acad. Sci. USA Vol. 73, 2191-21.
Page 95 (1976), MPWickens, GN Buell and RT Schimk
e, J. Biol. Chem. 253, 2483-2495 (1978), GMWah.
l, RA Padgett and GR Stack, J. Biol. Chem., Volume 254,
The method of pages 8679-8689 (1979) can be used to obtain copy DNA (cDNA). The RNA portion can be sequenced by chain cleavage known in the art using any of the methods described above, or by heat denaturation according to the method of Wickens et al. (1978).

Secondly, enzymes such as E. coli DNA polymerase I or AMV reverse transcriptase are described in the above references and JI Gordon, ATH Bur.
ns, JLChristmann & R.G.Deeley, J.Biol.Chem.Vol. 253,
CDNA is double-stranded DNA using the method of pages 8629 to 8639 (1978).
Can be used to change to.

Thirdly, synthetic linkers are, for example, Hind III or EcoR1
By using synthetic oligonucleotide linkers such as RHScheller, TLThomas, ASLee, WHKlei
n, WDNiles, RJ Britten and EHDavidson, Science
Vol.196, pp.197-200 (1977), TH Fraser and BJ Bru
ce, Proc. Natl. Acad. Sci. USA Vol. 75, 5936-5940 (197
8), A.Ullrich, I.Shine, J.Chivgwin, R.Pictet, E.Tische
r, WJRutter & H.M.Goodmann, Science Volume 196, 1313 ~ 13
Page 19 (1977), J. Ssine, PHSeeburg, JAMartial, JDB
axter & H.M.Goodmann, Natiure Volume 270, 494-499 (197
7), or PHSeeburg, J.Shine, JAMartial, JDBax
ter & H.M.Goodmann, Nature Vol. 270, pp. 486-494 (1977)
It can be ligated to both ends of double-stranded DNA using the usual methods described in.

In the fourth step, the DNA molecule is bound to the chromosome or is a plasmid, virus or cosmid known in the art.
smid), which may be a vector. Such vectors include:

pBR322 (F.Bolivar, RL Rodriguez, PJGreehe, MCBet
lach, HLHeyneker, HWBoyer, JHGrosa, S.Falkow, 197
7, Gene, Vol. 2, pp. 95-119) pMB9 (RL Rodrigues, F. Bolivara, HMGoodman, HWBoy
er, MCBetlach “Molecular Mechanisms in the Contro
l of Gene Expression "[DP Nierlich, WJRutter, C.
F. Fox, Eds.] P. 471, Academic Press, New York, 1976) pSC101 (SNCoher, ACYChang, HWBoyer, RBHellin
g, 1973, Proc. Nat. Acad. Sci. USA, 70, 3240) λgt WES (D. Tiemeier, L. Enquist, P. Leder, Nature 263)
Volume, 526-527) (1976) λcharo FUAGE (FR Blattner et al., Science, Volume 196, 161)
~ 169) (1977) f1R229 (JDBoeke, Molec.Gen.Genetics, Vol. 181, 288 ~
291) (1981) pJC75-58 (J. Collins, Methcd in Enzymology 68, 30.
9-326) (1979) This step is repeated outside the last host cell. Useful techniques for this method are described in the references above in connection with linkers, as well as the following references.
V.Hershfield, HWBoyer, C.Yanofasky, MALovett & K.M
urray, Nature, Vol. 251, pp. 476-482 (1974), FRBlattn.
Er et al., Science, 196, 161-169 (1977).

In the fifth step, the recombinant DNA molecule is, for example, M. Mandel & A.H.
iga (1970) J. Mol. Biol. 53, 159-162, PCWensin
k, DJ Finnegan, JE Donelson & D.S.Hogness, Cell 3rd
Volume, Pages 315-325 (1974), SNCohen, ACYChang and
L. Hsu, Proc. Natl. Acad. Sci. USA Vol. 69, 2110-2114,
(1972), HM Goodman, and RJ MacDonald, Methods i
n Enzymology 68, 75-90 (1979), EM Lederberg
And SN Cohen, J. Bact. 119, 1072-1074 (197
It can be introduced into the cytoplasm of the host cell line using the usual methods described in 4).

Accurate confirmation of clones requires hybridization selection (hybridization se
lection) or by probing with synthetic oligonucleotides (T. Taniguchi, Y. Fujii, Kuriyama and
M.Muramatsu, Proc.Natl.Acad.Sci.USA Vol. 77, 4003-400
Page 6 (1980), RPRicciardi, JSMiller & B.E.Roberts,
Proc. Natl. Acad. Sci. USA Vol. 76, 4927-4931 (1979),
DLMontgomery, BDHall, S. Gillam and M. Smith, Cell
14: 673-680 [1978]).

The newly modified host cells are then cloned and the expression of the desired material is obtained. For example, the technique of Guarente et al. (1980) using the lactose operon promoter (L.
Guarente, G.Lauer, TM Roberts & M.Ptashne, Cell No. 20
Volume, pp. 543-553 [1980], L. Guarente, TM Roberts & M. Pt.
ashne, Science 209, 1428-1430 [1980]), the expression of foreign DNA is obtained and optimized. Other promoters can be used to obtain expression known in the art as long as they are active in the desired bacterial, yeast or other host cell. Such promoters include the E. coli tryptophan operon, or β-lactamase promoters, and the S. cerevisiae, uracil 3 or invertase promoters.

In a particular embodiment of the present invention, a recombinant E. coli strain producing pre-prorennin A, prorennin A or rennin A can be obtained as follows.

1. RNA Isolation Fresh stomach tissue from milk-fed calves was obtained from a local slaughterhouse. The mucous membrane of the abomasum was separated from the stomach wall and frozen in dry ice. 21 g of mucosal tissue was added to 50 mM Tris ° HCl (pH 7.
5), 8M guanidine HCl, and 1 mM dithiothreitol were added to 200 ml of a cooled buffer solution (10 ° C) and the mixture was disrupted by a mixer. Insoluble material was removed by centrifugation at 10,000 rpm for 12 minutes in a Sorvall SA-600 rotator. 200 ml of surfacing material from the spin was added to 100 ml of chilled absolute ethanol. After 1.5 hours at -20 ° C, the pellet was pelleted by centrifugation at 3000 rpm for 30 minutes at -10 ° C. The pellet is 20 mM EDTA (pH
7), 20mM NaOAc (pH7), 8M guanidine HCl, and 1mM dithiothreitol ice-cooled buffer (EGAD)
It was dissolved in 40 ml. 20 ml of chilled absolute ethanol was added, then the solution was left at -20 ° C for 45 minutes. The precipitate was pelleted by centrifugation at 3000 rpm for 20 minutes at -10 ° C. The pellets were dissolved in 40 ml of cold EGAD buffer, precipitated with 20 ml of cold ethanol, centrifuged, and redissolved in pellets in EGAD buffer, which was repeated two more times. Finally, add a pellet to 20mM EDT
It was dissolved in 16 ml of A (pH 7) and extracted three times with chloroform: isobutanol (4: 1). Then 2 volumes of 4.5M NaOAc (pH 5.2) were added to the aqueous layer and the solution was left overnight at -20 ° C. The RNA precipitate was collected by centrifugation at 10,000 rpm for 25 minutes at -10 ° C and then dissolved in 30 ml of water.
The yield was 45 mg of RNA. RNA was precipitated by the addition of 1 ml of 2M NaOAc (pH 5) and 75 ml of absolute ethanol and then incubated overnight at -20 ° C. Pellet the RNA by centrifugation (10,000 rpm, 10 minutes, -10 ° C),
Redissolve in 20 ml of water, heat at 60 ° C for 10 min, cool rapidly on ice, and concentrate 2x concentrated binding buffer [20 mM Tris ° HCl (pH 7.
5), 2 mM EDTA (pH 7), 0.4% SDS and 0.24 M NaCl]. RNA was applied to a 4 ml oligo-dT-cellulose column, the column was washed with 1x concentrated binding buffer and then the poly A-containing RNA was eluted by washing the column with NaCl-free binding buffer. did. About 1 mg of poly A-containing RNA was obtained. Rabbit reticulocyte lysate
System (HRBPelham and REJackson [1976] Eu
rJBiochem. 67, 247-256). Protein products were analyzed on a 10% polyacrylamide gel. A single major protein band was observed, which precipitated with the rennin antiserum. This indicates that rennin mRNA is present in poly A-containing RNA.

2. Preparation of double-stranded copy DNA (cDNA) 50mM Tris ° HCl (pH8.3), 100mM KCl, 8mM MgCl ₂ , 0.4mM
20 ug containing dithiothreitol, 1 mM deoxynucleoside triphosphates, 100 units reverse transcriptase each
/ ml oligo (-dT) _12-18 and 1 Ci / mM α ³² P-dCTP were incubated at 42 ° C for 1 hour to obtain about 8.7 µg of cDNA from 20 µg of calf gastric poly A-containing RNA. Was synthesized. The reaction mixture was heated at 100 ° C for 3 minutes, then cooled on ice for 3 minutes, the precipitated protein was removed by centrifugation, and 100mM HepesKOH (pH6.9), 5m was added to half of the surface floating material.
M MgCl ₂ , 0.5 mM dithiothreitol, 0.125 mM deoxynucleoside triphosphonate were added. This mixture was mixed with 300 units of E. coli DNA polymerase I at 2 ° C at 16 ° C.
Double-strand cDNA 8.
6 μg was made. This DNA was extracted with phenol and
hadex G-100 (12ml column, 0.7cm × 30cm, 20mM Tris ° H
Cl (pH 7.5), eluted with 0.5 mM EDTA (pH 7.5)) and separated from the unincorporated triphosphite by chromatography, 2M NaOAc (pH 5) 1/10 volume , And ethanol precipitation at −20 ° C. overnight by addition of 2.5 volumes of chilled ethanol. Double-stranded cDNA (4.
6 μg) then 1 unit with 1000 units of Sl nuclease at 37 ° C
Time, Buffer S (0.3M NaCl, 30mM NaOAc (pH4.6), 3mM Zn
SO ₄ ). Reactions are 10 mM EDTA, and 200 mM Tri
It was terminated by the addition of s ° HCl (pH 8.3), the mixture was equilibrated over a Biogel A-150m column (0.7 cm x 33 cm), 10 mM Tris ° HCl (pH 7.5), 1 mM EDTA and 250 mM N.
Elute with aCl. Peak fraction of high molecular weight DNA (0.5 ml each)
Were pooled and ethanol precipitated by the addition of 1/10 volume of 2M NaOAc (pH 5) and 2.5 volumes of chilled absolute ethanol.

3. Addition of Hind III linker Double-stranded cDNA (1.7 μg) treated with Sl was added to Buffer T (25 mM T
ris HCl (pH8), 6.6 mM MgCl ₂ , 0.5 mM EDTA, 5 mM _2- mercaptoethanol and 0.5 mM each of deoxynucleoside triphosphates) with 2 units of T ₄ DNA polymerase at room temperature for 30 minutes. did. This material was extracted with phenol, then ether, and ethanol precipitated by addition of 1/10 volume of 2M NaOAc (pH 5) and 2.5 volumes of ethanol. This double-stranded cDNA with blunt ends was then labeled with 66 mM Tris ° HCl (pH 7.6),
Hind III synthetic linker labeled with ³² P in 6.6 mM MgCl ₂ , 5 mM _2- mercaptoethanol, 0.5 mM ATP 300 pmol (10 pM)
Incubation was carried out overnight at 12 ° C. with 0 × excess cDNA ends) and 9 units of blunt-ended T ₄ DNA ligase.

The reaction was adjusted with 10 mM EDTA (pH8), Biogel A-150m
Fractionation was performed on a column (0.7 cm x 20 cm). Fractions containing high molecular weight DNA (0.25 ml each) were pooled and ethanol precipitated. Restrict this material to the Hind III endonuclease (9
Unit), 5.6mM Tris ° HCl (pH7.6), 5.6mM MgCl ₂ 37
It was treated at 45 ° C. for 45 minutes, then extracted with phenol, extracted with ether and ethanol precipitated by addition of 1/10 volume of 1M NaOAc (pH 5) and 2.5 volumes of absolute ethanol. This double-stranded cDNA with Hind III sticky ends was then ligated to fl Phage CGF4 double-stranded DNA. This fl-fusion CGF4 double-stranded DNA was cleaved and cleaved with a restriction Hind III endonuclease, and the method of H. Goodman and RJ MacDonald (HM Goodman and RJ MacDonald [1979], Methods i
n Enzymology 68, 75-91) to remove terminal phosphates by two treatments with calf intestinal phosphatase {Note: to generate pharage CGF4, fl Fage R229 (JD Boecke [1981] Mol.Ge
n.Genet. 181, 288-291) was cleaved with EcoRI endonuclease and blunt-ended with T ₄ DNA polymerase.
It was then attached with a Hind III synthetic oligonucleotide linker from Collaborative Research, Inc. (Waltham, Massachusetts)}. 66 mM Tri for this binding reaction
s ° HCl (pH7.6), 5mM 2-mercapto-ethanol, double-stranded
cDNA 0.3 μg, CGF4 DNA 0.2 μg, 0.5 mM ATP and sticky ends 30
It contained 0 units of T ₄ DNA ligase. Binding reaction at 16 ° C
It was held for 29 hours.

4. transflector Ekushi Yong of E. coli BNN45 with recombinant -CGF4DNA (transfection) E. CGE6 strain ^{(BNN45; hsdR -, hsdM +} , supE, supF, Bl -, me
t ^-) were grown with shaking at 37 ° C. in a tryptone (tryptone) Biiyon, 10 min at 4 ° C., and harvested by Yotsute OD ₇₀₀ = 0.5 to centrifugation at 7000 rpm. The cells were resuspended in ice-cold 50 mM CaCl ₂ (1/2 of the original culture volume) and then left at 0 ° C. for 30 minutes. Suspension then 4 ° C
Centrifuge at 7,000 rpm for 10 minutes at 50 mM CaC cooled with ice
Suspended in ₁₂ (1/20 of original culture volume). After standing at 0 ° C. for 60 minutes, the cells were used for transfection. 1 of 20μ ligation reaction
/ 2μ was added to each of eight test tubes containing 50μ of sterile 50mM Tris HCl (pH 7.6). Add 1/10 ml of cells treated with CaCl ₂ to each tube and mix the mixture on ice.
Let stand for a minute. After heating to 37 ℃ for 2 minutes, CGE5 (JM101:
J.Messing ^{[1979], V (lacpro) SupEthi - F'tra} D36 pro AB lac IZVM15) overnight culture in-back ground
0.2 ml and 0.7% soft agar (agar) were added and the mixture was poured onto 8 tryptone agar plates. 37 ° C
Incubating overnight at about 250 plaques per plate.

5. Identification of recombinant CGF4 carrying the rennin coding sequence These plaques were transferred to nitrocellulose and ▲ ³² _α ▼
P-dCTP and reverse transcriptase (TPSTJohn and RWDa
The method of Benton & Davis (WD Benton and RWD) using a ³² P-labeled cDNA made from poly A-containing PNA of calf stomach using vis [1979] Cell 16: 443-452).
avis [1977] Soience 196, 180-182). Approximately 80 highly recombinant recombinant phages to the labeled cDNA were picked from the plates and stored in TY medium at 4 ° C. An intact (complete) forage sample was expanded by overnight growth on CGE5 cells, harvested by centrifugation, then 2% agarose containing 0.37M Tris ° g glycine (pH 9.5). Electrophoresed in gel, treated with 0.2N NaOH for 1 hour and 0.5 MT
Ethidium bromide (ethid bromide) after neutralization in ris ° HCl (pH 7.4)
ium). The migration is inversely proportional to the size log of the phage DNA, and 8 phages with inserted DNA of 1000-2000 base pairs in size were selected. Double-stranded RFI DNA was analyzed by the method of Moses et al.
B. Moses, JD Boeke, K. Horiuchi & N.D.Zinker (1980) Vi
rology, Vol. 104, p. 267). This DNA was cleaved with Hind III and the resulting fragment was analyzed on an agarose gel to verify that the insert was in the Hind III site and of the expected size. did. Finally, DNA from 4 recombinant phages (about 5-10 μg from each)
And the DNA from the vector CGF4 was cut with Hind III and the fragments were denatured by boiling for 45 minutes and after freezing in dry ice / ethanol the DNA in water was
Attached to nitrocellulose by spotting onto a piece of dried nitrocellulose pretreated with 20 × SSC. After baking under vacuum at 75 ° C for 1.5 hours,
The hybrid selection method described by Miller et al. For DNA bound to nitrocellulose (JSMill
er, RPRicciardi, BERoberts, BMPaterson & M.B.Mat
hews [1980] J. Mol, Biol. 142, 455-488), and RNA2 of calf stomach enriched with poly A for each hybridization.
μg was used. The eluted RNA was analyzed by the method of Pelham and Jackson (HRBPelham R.J.Jackson [1976] Eur.J.Bioche.
m. 67, 247-256). Reticulocytely lysate labeled with ³⁵ S-methionine.
sate) system and translate the resulting protein product into Laemml
Method i (U. Laemmli [1970] Nature Vol. 227, 680-685
Page) on a 10% polyacrylamide gel containing 0.1% SDS. The results of the gel analysis showed that all four phage DNAs tested showed a protein product identical to pre-prorennin in post-translational size and immunological criteria in rabbit, reteikiurosite, and lysates. The resulting RNA species was selected, indicating that the phage DNA was hybridized to rennin mRNA. 2 out of 4
293-207 with an insert of about 1400 base pairs (bp) and 293-118 with an insert of about 1250 bp.
/ 37 was selected for further investigation. Maxa DNA insert
m and Gilbert (AM Maxam and W. Gilbert [1980] Me
thods in Enzymology 68, 499-560). Nucleotides 205 to 1350 are the DNA sequence for the pre-prorennin A gene (see Table 1). Nucleotide sequences 1-204 and 1351-1460 are linked to pre-prorennin, but can be removed if desired and are not essential for the use of the gene in expression. Useful portions of the DNA material of Table 1 can be isolated and used according to known techniques.

This table combines information from both 293-207 and 293-118 / 37. That is, recombinant phage
293-207 includes nucleotides # 1 to at least nucleotide # 13, in addition to the deleted nucleotides 848 to 961.
Up to 60 have an insert with the sequence shown in Table 1, while Phage 293-118 / 37 has an insert with the sequence from nucleotide # 229 to nucleotide # 1460. As revealed by the sequence results, initiation of rennin synthesis occurs at the methionine codon (nucleotides 205-207), resulting in a pre-prorennin molecule with 16 additional amino acids compared to purified prorennin. (Prorennin B amino acid sequence is B. Foltmann et al., Proc. Nat. A
cad.Scl.USA Vol.74, pp.2321-2324 (1977) and B.Fol.
Tmann et al. J. Biol. Chem. 254, 8447-8456 (1979). The nucleotide sequence data in Table 1 is the first indication of the presence of pre-prorennin. )
The two recombinant fl phages 293-207 and 293-118 / 37 have the DNA sequence for the entire pre-prorennin A molecule when combined together. Pre-prorennin A
Has a different prorennin moiety at amino acid # 290 from prorennin B (as described by Foltmann et al. (See above) in aspartate in rennin A and glycine in rennin B;
Of that). The codon for asparagine is shown at amino acid position # 204, while Foltmann reported aspartate at that position. However, since the aspartate and glutamate amides are difficult to distinguish from their acid forms, while the nucleotide sequences can easily distinguish the codons, the above report misleads the amino acid sequence. It seems to be.

The cloned rennin gene, designated Fage 293-118 / 37, was used to study the properties of bovine genomic or rennin gene copies. These experiments
Notch (nick) -Method of translation (PWJRigby, M. Diekman
n, C. Rhodes, and P. Berg [1977] J. Mol. Biol. Vol. 113, 2
37-251), the 32P-labeled cloned rennin DNA is hybridized to be cleaved with various restriction enzymes and separated on an agarose gel, followed by the Southern method (EMSouthern [1975] J. Mol. .Biol. Volume 98, 503-51
P. 7) on bovine DNA transferred to a nitrocellulose membrane. The results show that restriction endonuclease cleavage of bovine DNA by enzymes such as Sac I and Bg / I (which does not cleave cloned pre-prorennin cDNA sequences) nevertheless hybridizes to rennin sequences. It has been shown to produce one or more bands of DNA. This means that (a) the genomic copy of the rennin information contains additional DNA (possibly an intervening sequence) containing restriction enzyme sites not found in the rennin cDNA, or (b) one or more rennin. The gene is present in the genome, suggesting that some restriction enzymes cut between the copies. This latter possibility was eliminated by hybridizing restriction-cut bovine genomic DNA with 32P-labeled probes obtained from the 5'and 3'ends of the cloned rennin cDNA. These results are consistent with a single genomic copy of the rennin-encoding information using the restriction endonucleases EcoRI and BamHI, for example. This means that the A and B forms of rennin observed by B. Foltmann et al. (J. Biol. Chem. 254, 8447-8456 [1979]) generate two different alleles of the rennin gene. It means very similar to things. Furthermore, the bovine genomic copy of the rennin gene contains intervening sequences, and in that respect it differs from our cloned cDNA which is identical to the messenger RNA for pre-prorennin.

6. Expression of pre-prorennin in E. coli A plasmid, pCGE5, designed to facilitate the expression of pre-prorennin in E. coli is a three segment of agarose gel purified DNA.
It was composed by. Plasmid pBR322 (4.5 μg) was treated with restriction endonucleases, Hind III (NEBiolabs, 6 units) and Pst I (NEBiolabs, 3 units) at 37 ° C. for 1 hour, 50 mM NaCl, 7 mM Tris ° HCl (pH 7.5), 7 mM MgCl 2. Cleavage was carried out in 50 μreaction containing ₂ and 6 mM 2-mercaptoethanol. Recombinant Forge 29
Double-stranded RFI DNA (3-μg) from 3-207 was treated with restriction endonuclease Kpn I (NEBiolabs, 10 units) for 1 hour at 37 ° C, Buffer K (6mM NaCl, 6mM Tris ° HCl (pH7.5), 6m).
M MgCl ₂ , and 6 mM 2-mercaptoethanol) in 50 μl of the reagent. Plasmid pLG400
(L. Guarente, G. Lauer, T. Roberts, M. Ptasne, Cell No. 20
Vol., Pp. 543-553, 1980).
(NEBiolabs, 6 units) at 37 ° C for 1 hour in Buffer H (6 units)
Cleavage was carried out in 50 μg of reagent containing 0 mM NaCl, 7 mM Tris HCl (pH 7.5), 7 mM MgCl). Extracted with phenol, extracted with ether, and after the ethanol precipitation were line summer, cut DNA from 293-207 and pLG400 in order to create blunt ends at the restriction cleavage, T ₄ DNA polymerase (P-L Biochemicals, 10 units) at 37 ° C
30 minutes, Buffer T (25mM Tris ° HCl (pH8), 6.6mM MgC
l ₂ , 5 mM 2-mercaptoethanol, 0.5 mM EDTA, 0.5 each
mM deoxynucleotide triphosphate) was separately treated in 50μ of reagent. After phenol extraction, ether extraction and ethanol precipitation, pLG400DNA was further Pst I (NEBiolabs, 3 units).
Buffer p (50 mM NaCl, 7 mM Tris ° H) at 37 ° C for 2 hours.
Cl (pH 7.5), 7 mM MgCl ₂ 6 mM and 2-mercaptoethanol) 50 μ, while 293-207 DNA is Hind
III (NEBiolabs, 6 units) at 37 ° C for 2 hours, BufferH5
Cleaved in 0μ. Each of the three preparations of restriction digested DNA was extracted with phenol, extracted with ether, ethanol precipitated, redissolved in 30 [mu] of water and loaded on a preparative, horizontal 1% agarose gel. After gel electrophoresis in 40 mM Tris acetate (pH7.2) at 70-80 volt for 3-4 hours, the gel was washed with ethidium bromide (ethidiu bromide).
m) and stained under long wavelength UV light. 293-207
500 base pair (pb) band from, pLG400 to 6000b
Cut out the p band and the 800 bp band from pBR322,
DNA by freezing and thawing pieces
Was extracted (Thuring et al., Anal. Biochem. 66, 213 [1975]). All three DNA segments were ethanol precipitated and redissolved in water. About 0.15 of each piece
Buffer mol (66 mM Tris ° HCl (pH 7.
5), 6.7 mM MgCl ₂ , 10 mM dithiothreitol, 0.75 mM A
TP) and T ₄ DNA ligase (NE Biolabs, was 600 units) were coupled together in the reactant 20μ containing. A cell of Escherichia coli CGE6 strain having a conversion ability was prepared in the same manner as the method described in the above section 4, and 50 mM Tris · HCl (pH 7.6) 50 μm was added.
The bound DNA (5 μm) therein was mixed with 100 μm of the cells at 0 ° C. for 1 hour, heat-treated at 37 ° C. for 2 hours, and then diluted 10-fold with fresh trypton broth. After incubation at 37 ° C. for 1 hour with shaking, the cells were plated on tryptone plates containing 20 μg / ml ampicillin. Ampicillin resistant colonies were selected, plasmid DNA was prepared and analyzed by restriction enzyme digestion. Several of these standard strains yielded the desired plasmid pCGE5 shown in FIG. Analysis of the DNA sequence revealed that the binding between 293-207 DNA and pLG400 DNA was expected. Therefore, the 5'end of the pre-prorennin is fused to the 3'end frame of the I "Z fusion of Guarente et al. Plasmid DNA is standardized (DB Clewell and DR Helinski, Proc. Nat. Acad. Sci.
USA. 62, pp. 1159-1166 [1969]).

The DNA fragment responsible for the lactose operon promoter and ribosome binding site contained 10 μg of DNA in Pvu II (NE
Biolabs, 7.5 units) and Pst I (NEBiolabs, 4 units)
Plasmid pGL101 (L. Guaren by digestion at 37 ° C. for 2 hours in 100 μl Reagent containing Buffer P.
te, G. Lauer, T. Roberts and M. Ptashne, Cell Vol. 20, 54
3-553 [1981]). The 850 bp fragment was isolated from a preliminary agarose gel by the strip removal and freeze / thaw techniques described above.

Plasmid pCGE5DNA (40 μg) was prepared using Hind III (CRI, 70
Units) at 37 ° C. for 1 hour in 150 μl of Reagent containing Buffer H (see above). This DNA is then exonuclease Bal31 (NEBiolabs, 5 units) at 10 ° C for 10
Min, Buffer B (0.6M NaCl, 12mM CaCl ₂ , 12mM MgC
Digested in 200 μl of a reaction medium containing l ₂ , 20 mM Tris ° HCl (pH 8), 1 mM and EDTA) (see FIG. 2). Analysis by gel electrophoresis showed that Bal31 treatment removed a sufficient number of nucleotides resulting in a group of fragments with a 5'end near the ATG start codon for pre-prorennin. The DNA was made blunt-ended by the method described above using T ₄ DNA polymerase, then the DNA (1 μg) was Pst I.
(NE Biolabs, 0.06 units) at 37 ° C. for 5 minutes, partially digested in 20 μl of Reagent containing Buffer P. Following this DNA is, T ₄ DNA ligase in Buffer L (CRI, 300 units)
Ligation was carried out in 20 μl of a reaction solution containing lignin with 850 bp of a DNA fragment having a lactose operon promoter and a ribosome binding site (0.2 μg). Cells capable of converting Escherichia coli CGE7 strain (NK5031, su III ⁺ , lacVM5265, nal
r, F ⁻ , Bl ⁻ ) was produced in exactly the same manner as described above for strain CGE6, 100 μl of cells in suspension were transformed with 5 μl of said reagent, incubated and subjected to heat shock, It was grown for expression of the ampicillin resistant phenotype in exactly the same manner as described above for the conversion of CGE6 by pCGE5. Cells were plated on MacConkey lactose and ampicillin (20 μg / ml) medium. Dark red colonies showing β-galactosidase were selected,
The β-galactosidase activity was evaluated and analyzed (J. Miller, E.
xperiments in Molecular Genetics, New York, Cold Spring Harbor Laboratory, 197.
2). Plasmid DNA was transferred to 12 of these transformed cells (transf
ormant) and analyzed by digestion with restriction enzymes and electrophoresis on agarose or polyacrylamide gels. One strain, CGE20, is plasmid pCGE17 (Fig. 3
See above) Approximately 40 nucleotides from the ATG start codon of the pre-prorennin-I ″ Z fusion above, the lactose operon promoter, and intermediate levels of β-galactosidase (fully induced in lactose ⁺ strains such as CGE6). About one-third of the level).

To create a plasmid with the entire pre-prorennin gene fused to the lactose promoter and the ribosome binding site, pCGE17DNA (4 μg) was added to Bgl II.
(NE Biolabs, 6 units), cut in 90 μl of Reagent containing Buffer P for 1 hour at 37 ° C. Then, 100mM Tr
is ° HCl (pH 7.5) was added, and the DNA was further converted into EcoR I (Boe
hringer / Mannheim, 40 units), and cut at 37 ° C for 1 hour. Next, pBR322 DNA (5 μg) was added to Pvu II (NEBiolab
s, 5 units) at 37 ° C. for 1 hour in 45 μl of reagent containing Buffer P, and then 100 mM Tris ° HCl (pH 7.5)
And EcoR I (Boehringer / Mannheim, units)
Incubation was further performed at 37 ° C for 1 hour. Finally,
Hind recombinant fl Phage 293-118 / 37 RFI DNA (6 μg)
III (NEBiolabs, 6 units) at 37 ° C for 1 hour in Buffer
Cleavage was performed in 50 μl of H containing reagent. Cleaved DNA after phenol extraction and ethanol precipitation
With T ₄ DNA polymerase (PL Biochemicals, 10 units) at room temperature for 30 minutes, 50 μl of reaction reagent containing Buffer T
Hind III sites were smoothed by treatment in. Then,
Redissolved, phenol extracted and ethanol precipitated
DNA with Bgl II (NEBiolabs, 4 units) for 1 hour at 37 ° C,
Cleavage was done in 30 μl of Reagent containing Buffer P. Three restriction enzyme digested DNA species were smeared on a preliminary horizontal agarose gel, then pCGE17 fragment 370 bp, pBR322 fragment 2300 bp and 293-118 / 37 fragment 1000 bp were excised and the agarose mass was frozen and thawed. Eluted. After performing ethanol precipitation, the DNA was redissolved in water, and about 0.2 pmol of each fragment was added to 20 µl of a reagent containing Buffer L and T ₄ DNA ligase (NEBiolabs, 300 units) for 6 hours at 14 ° C.
Bound to each other in. The E. coli CGE6 strain was transformed with the ligated DNA by the method described above, and ampicillin resistant colonies were selected. Analysis of the plasmid DNA by restriction enzyme digestion revealed that the CGE24 strain harbors the plasmid pCGE21 (see FIG. 4) containing the entire pre-prorennin sequence fused to the lactose operon promoter and ribosome binding site. Natsuta.

Two analyzes indicate that this strain is authentic synthetic bovine pre-prorennin. First, the crude extract of the cells was determined by the method of Peak et al. (GJPeak, J. Morris and MJB).
uckman (1979) “Growth Hormones” in Methods of Horm
one Radioimmunoassay, pages 223-244 (BMJaffe & H.R.Be
hrman] In the radioimmunoassay performed by Academic Press, New York), the binding of iodinated rennin to anti-rennin serum is inhibited. However, the following modifications are made to the above method. That is, iodinated rennin is 50 mM sodium phosphate (pH 6) 0.15 MN
Stored in aCl, 1% BSA, RIA buffer was 0.05M Tris ° H
Consists of Cl (pH8), 0.5% human serum albumin and 0.1% sodium nitrite.
Done on time. The amount of inhibition was about 0.8 for pre-prorennin.
.mu.g is present in each ml of extract (or approximately 4 .mu.g of pre-prorennin per cell culture). Second, cells are mid-exponential.
pulse for 30 minutes in phase) with 35S-methionine.
e) Labeled, lysed and immunoprecipitated with anti-rennin serum. (JS Emtage et al Nature 283, 171-175 [198
[0]. ) SDS (UKLaemml)
i & M. Favorite, J. Mol. Biol. 80, 575-599 (1973))
Analyzed on a 10% polyacrylamide gel containing
When autoradiography was performed, a band having the same size as that of pre-prorennin was observed. In addition, a band of the same size as rennin was also observed, which indicates that the bacterium is converting pre-prorennin to rennin or that a second initiation of translation occurs in the pre-prorennin sequence. It is a suggestion. No band was present in the immunoprecipitation of the plasmid-free parent strain CGE6. These results indicate that pre-prorennin is produced in E. coli cells carrying the plasmid pCGE21 and the level of production is approximately 600 molecules per cell. High levels of production can be achieved by using a similar scheme to obtain a fusion of the lactose operon promoter close to the start codon of pre-prorennin.

The extract of CGE24 strain was also analyzed by B. Foltmann's standard milk-coagulation assay (Methods in Enzymology [1970] Vol. 19, 421-43).
Activity on page 6). The results show that this E. coli strain produces about 100 molecules of active rennin per cell or an active fragment of rennin capable of coagulating milk, whereas an extract from the CGE6 strain, which does not contain a rennin DNA sequence, coagulates milk. It shows that you cannot do it. The CGE24 strain containing the plasmid pCGE21 is American
Deposited under the accession number 31929 at the Type Culture Collection (ATCC).

The CGE24 strain is a plasmid pCGE2 defined according to FIG.
Escherichia coli BNN45 strain having 1 (hsdR-hsdM + supE 44 supFB
l ^- the met ^-) is (Advanced Bacterial Jieneteitsukusu, RWDavis, D.Botstein, JRRoth, Cold Spring Harbor Laboratory, Cold Spring Harbor, Niyuyoku, pp 1980,7). This plasmid is the plasmid pBR322 (nucleotide 2067
~ 4360, JGSutcliffe (1979) Cold Spring
Harbor Symposium, Vol. 43, p. 77-90), as well as EcoRI and plasmid pGL101 (L. Guarent).
e, G. Lauer, TM Roberts and M. Ptashne [1980] Cell No.
20, p.543-553) by means of the Pvu II site (recombinant fl Phage 293-207 and
(From 293-118 / 37) containing a fragment of 95 base pairs fused to approximately 1300 nucleotides. The arrangement is such that the lactose operon promoter drives the expression of the pre-prorennin protein in E. coli (see Figure 5).

7. Expression of methionine prorennin in E. coli The pre-prorennin gene is a restriction endonuclease.
It contains three acceptable sites for Hha I (recognizing GCGC [see Table 1]), one of which removes the "pre" coding sequence and the first for the alanine codon from the sequence for prorennin. Leave the nucleotide G removed. Therefore, we isolated this partial Hha I digestion product, which represents an almost intact (complete) prorennin gene. RFI2 from recombinant phage 293-118 / 37
18 μg of double-stranded DNA was digested with 12 units of restriction Hind III (NEBiolabs) in 50 μm of Buffer H at 37 ° C. for 1 hour. About 1230 bp insert with rennin DNA was purified by extracting the DNA from the appropriate band on a 1% agarose gel by the freeze / thaw method. Buff about 1.5 μg of this DNA
3 with 0.25 units of Hha I (NEBiolabs) in er P 30 μ
A partial Hah I cut was made by incubating for 5 minutes at 7 ° C. DNA corresponding to the uncleaved fragment plus the single-cleaved fragment lacking approximately 25 nucleotides from the start of 293-118 / 37 was isolated from the band on a 2% agarose gel. Plasmid pBR322
Restrict the endonuclease Hind II (NE
Biolabs, 9 units) in Buffer H 100μ for 1 hour at 37 ° C. This DNA was phenol extracted and then ethanol precipitated. Approximately 0.5 p each of DNA (ie partially Hha I cut 293-118 / 37 and Hind III cut pBR322)
Moles were combined, redissolved in 28μ of water, and then Buffe with DNA polymerase I (Boehringer / Mannheim, 9 units).
Reagent 40μ containing r D (60mM Tris ° HCl (pH7.5), 80mM MgCl ₂ , 10mM dithiothreitol, 1mM ATP and 0.2mM deoxynucleotide triphosphonate)
The ends were blunted by treating at 10 ° C for 10 minutes. A synthetic oligonucleotide containing the Xba I restriction endonuclease sequence plus ATGG (ie, CCATCTAGATG
G) was synthesized by the triester method by Collaborative Research (K. Itakura et al., J. Biol. Chem. Vol. 250, p. 4592 [1975]), and 5 μg thereof was synthesized using T ₄ polynucleotide kinase (P). -L biochemicals) 6 units using Buffer Y (70 mM Tris HCl (pH 7.6), 10 mM MgCl
_{It was} kinased with α ³² -P-ATP in 25 μl of a reagent containing 210 mM 2-mercaptoethanol and 2 mM ATP). Add buffer components and T ₄ DNA to keep the 5'-labeled oligonucleotide, constant its concentration
Blunt end reagent 40 μ was added along with 600 units of ligase (NEBiolabs). The reagents were incubated overnight at 14 ° C, then 180 mM NaCl, 7 mM MgCl ₂ and 5 mM Tri.
Diluted with 5 volumes of a solution consisting of s ° HCl (pH 8). After heating at 65 ° C for 5 minutes, the DNA was treated with 45 units of Xba I restriction endonuclease (1 for a total digestion time of 3 hours).
15 units were added per hour). Finally, the oligonucleotide monomer was loaded onto a Bio-Gel A-5m column (0.68 x 36c).
m, see above) large DNA by gel passing over
Separated from. Excluded DNA was pooled, ethanol precipitated, redissolved in 12 μl of water and Buffer L and T ₄ DN.
Incubation was performed overnight at 14 ° C. in a coupling reagent containing 300 units of A ligase (NEBiolabs). 5 μl of this binding reagent was used to impart the conversion ability to cells of the CGE6 strain as described above. The transformed cells were plated on tryptone plates containing 20 μg / ml ampicillin,
Colonies were selected and screened for etracycline sensitivity. Plasmid DN by restriction enzyme digestion (Xba I plus Kpn I) and polyacrylamide gel electrophoresis
Analysis of A shows that the desired plasmid pCGE181 (ie Xba
1 strain (resulting in an I-Kpn I fragment).

Approximately 5 μg of pCGE181 DNA with Xba I (NEBiolabs, 4 units) 37
Buffer X (150mM NaCl, 6mM Tris ° HCl (p
H7.9), 6 mM MgCl ₂ ). After phenol extraction and ethanol precipitation, the DNA was treated with T ₄ DNA polymerase (PL Biochemicals, 10 units) for 30 minutes at 37 ° C. in 50 μl of a reagent containing Buffer T. The blunt end. Again, the DNA is phenol extracted and then ethanol precipitated. Vector DNA is plasmid pGL101DNA
(L. Guarente et al. Cell Vol. 20, pp. 543-553 [1980]) 5 μg
With Pvu II (NEBiolabs, 5 units) by cleavage in 50 μl of Reagent containing Buffer P.
Then, after phenol extraction and ethanol precipitation, the redissolved DNA was treated with 0.06 units of calf intestinal alkaline phosphatase (Boehringer / Mannheim) at 37 ° C.
Phosphoatase by treatment in 50 μl of Reagent containing Buffer C for 30 min. The PvuII-cleaved vector (0.2 pmol) and the XbaI-cleaved prorennin DNA fragment (0.2 pmol) are ligated together at 14 ° C. overnight in 20 μl of Reagent containing Buffer L. Cells capable of converting the CGE6 strain are prepared as described above and converted with the binding reagent 5μ. The resulting cells are plated on tryptone agar plates containing 20 μg / ml ampicillin. Ampicillin resistant colonies are selected and plasmid DNA is isolated and analyzed by restriction enzyme digestion and agarose gel electrophoresis. A strain is found that has a prorennin DNA bound to the ribosomal binding site such that the lactose operon promoter and prorennin protein are made in vivo (ie, the ATG start codon added to the prorennin sequence is 9 lactose operon ribosomal binding sites). Of nucleotides).
We used the lysate of cells carrying the plasmid
The amount of prorennin synthesized is determined by subjecting it to radioimmunoassay using iodinated authentic purified rennin and anti-rennin serum. The size of the prorennin product is determined by electrophoresis of immunoprecipitates of ³⁵ S-methionine labeled cell extracts on polyacrylamide gels containing SDS.

8a. Expression of methionine-valine-rennin in E. coli To obtain DNA containing only the rennin coding sequence,
The RFI DNA from recombinant phage 293-207 was digested with nuclease Ba
It was excised with l31 and ligated into the fl phage vector. This ligation product was cloned and excised rennin
A DNA library was created. Specifically, 8 μg of 293-207 Phage RFI DNA was cleaved with 6 units of Hind III (NEBiolabs) in 20 μl of a reagent containing Buffer H at 37 ° C. for 1 hour. After performing phenol and ether extractions and ethanol precipitation, the DNA was redissolved in 20μ of water and treated with 1.25 units of Bal31 (NEBiolabs) in 50μ of reagent containing Buffer B for 30 minutes at 30 ° C. The reaction was stopped by phenol extraction and ethanol precipitation. Bal31 excised DN, ranging in size from 500 to 1000 bp
The A fragment was isolated from a 1.5% agarose gel by the freeze / thaw technique described above. The excised DNA was treated with DNA polymerase I (Boehringer / Mannheim, 9 units) in 40 μl of a reaction agent containing Buffer D at 10 ° C. for 10 minutes to make it a blunt end. A synthetic oligonuclease linker (specifically, Hind III 8-mer CAAGCTTG; manufactured by Collaborative Research Co., 5.0 μg) contains Buffer Y using 6 units of T ₄ polynucleotide kinase (PL Biochemicals). ³² P in 25μ of reagent
-Kinase-ized with ATP. The labeled oligonucleotide was added to 40 μl of blunt-ended reagent along with additional buffer components to keep its concentration constant and 600 units of T ₄ DNA ligase (NEBiolabs). The reagents were incubated overnight at 14 ° C. Next, the reaction agent was added to 60 mM NaCl.
And diluted 5 times with 250 μM of 7 mM MgCl ₂ and heated at 65 ° C. for 5 minutes. After cooling the reaction mixture, all 45 units of Hind III restriction endonuclease (NEBiolabs) were added in 3 portions of 15 units per hour and incubated at 37 ° C. for 3 hours. The oligonucleotide linker monomer was applied to a Biogel A-5m column (0.68 x 36 cm, BioRa).
d) Column buffer (10mM Tris ° HCl (pH7.5), 100m)
The mixture was removed by eluting in M NaCl, 1 mM EDTA). The excluded peak was ethanol precipitated, the DNA (about 0.5p moles), Buffer L, T ₄ DNA ligase (NE Biolabs) 600 units and had been Yotsute Fuosufuataze into cut and the method by Hind III phage CGF4 RFI DNA (Collaborative Genetics) was added to 20 μl of the binding reagent containing about 0.5 pmol. After binding at 14 ° C for 18 hours, 50 mM Tris ° HCl (pH 7.
4 µl of the reaction mixture diluted 4-fold in 6) was used to confer cells of the CGE6 strain on conversion capacity. The conversion ability and the plaque flattening were carried out by the same method as described in 4 above. About 500 plaques were obtained per plate. B
By the method of Enton and Davis (Science 196, 180-182 [1977]). Nick-translaed nick-translaed pre-prorennin DNA contained on plasmid pBR322 (PWJ Rigby et al. [1977] J. Mol. Biol. 113).
Vol. 237-251) showed about 15% of plaques containing rennin DNA.
Take about 250 of these and put them in tryptone broth at 4 ° C.
Stored in. Analysis of DNA from some of these recombinant phages by digestion with restriction enzymes and electrophoresis on an agarose gel revealed that the 5'end of the inserted sequence was at the start of the rennin-encoding sequence (ie, shown in Table 1). Figure 3 shows several phages with Bal31 excised DNA from pre-prorennin DNA to approach nucleotides 379) in the sequence. Single stranded recombinant fl phage DNA was isolated from these phage as follows. First, a slab of Phage was placed on a CGE5 overnight culture of 0.4 m.
Prepare by infecting l with 50μ of phage collected from plaque. Transfer this onto a tryptone agar plate in 7 ml of 0.7% soft agar. The phage is eluted by adding 12 ml of tryptone broth to the plates after overnight growth and incubating for 2 hours. Then 3 ml of the broth were precipitated with 0.6 ml of polyethylene glycol / NaCl solution (25% PEG and 2.5M NaCl) and stored for 1 hour at 4 ° C. (KRYamamoto et al., Virology Vol. 40, 734).
~ 744 [1970]). After centrifugation, the B is
uffer TEN (10mM Tris ° HCl (pH8), 10mM NaCl, 0.5mM
Resuspend in 0.3 ml of EDTA). Then, forge the PEG / Na
Precipitate with 30 μl of Cl solution, incubate at 4 ° C. for 1 hour, and centrifuge. Resuspend the phage in 50μ of Buffer TEN and add 5.5μ of 1% SDS to each tube. 55
After incubating at ℃ for 10 minutes, TEN200μ is added, and the solution is extracted with phenol, extracted with ether, and precipitated with ethanol. The sequence of the inserted dna in the recombinant fl phage was determined by the method of Sahger (F. Sanger et al., Mol. Biol.
Vol. 161-178 [1980]) using synthetic oligonucleotide primers ("Universal primers" with the TTGACGGGGAAAG sequence, Collaborative Research, Waltham, Massachusetts). At least one phage was found in the collection of the Bal31 excised insert cloned in fl,
(293-207-101), which is nucleotide 3
At 79 (ie at the codon for glycine, the N-terminal amino acid of mature rennin), is the Hind III linker fused to the 5'end of rennin.
The RFI DNA (5 μg) of Phage 293-207-101 was Hind III.
Digest with (4 units, NEBiolabs) for 1 hour at 37 ° C. in 50 μl of Reagent containing Buffer H. After performing phenol extraction and ethanol precipitation, the DNA was
Redissolved in nuclease Sl (Boehringer / Mannhei
m) 10 units, 37 ° C for 10 minutes, Buffers S (see 2 above)
Are treated in 50 μl of the reactant containing This DNA is again phenol extracted and then ethanol precipitated. Next, synthetic oligonucleotide linker (having CCATCTAGATGG sequence, 5 μg, Collaborative Research)
Is kinased with α- ³² P-ATP using 6 units of T ₄ polynucleotide kinase (PL Biochemicals) in 25 μl of Reagent containing Buffer Y. This kinased oligonucleotide is treated with Sl (see above) and ligated by freeze / thaw method onto the Hind III-conjugated insert which had been purified from agarose gel. The binding mixture is Hi
0.8 pmol of nd III-conjugated Sl-treated rennin DNA, 100 pmol of kinased synthetic linker and T ₄ DNA ligase (NEBiolab
s) 600 units are contained in 20 μl of Buffer L. 18 at 14 ° C
Incubate time. Reactant 180mM NaCl, 7m
A solution of M MgCl ₂ and 5 mM Tris ° HCl (pH 8) 6 at 100 μm
Dilute twice. After heating at 65 ° C. for 5 minutes, 45 units of restriction endonuclease Xba I (NEBiolabs) are added in triplicate, 15 units each during incubation at 37 ° C. for 3 hours. Oligonucleotide monomers are separated from large DNA by passing over a Biogel A-5m column (0.68 x 36 cm) in column buffer (see above). Ethanol precipitate the removed DNA and use Buffer T 50
Subject to treatment with T ₄ DNA polymerase (PL Biochemicals, 10 units) in μ to blunt its Xba-cut ends. After performing phenol extraction and ethanol precipitation, the DNA was buffer R (100 mM Tris ° HCl (pH 7.
5), 50mM NaCl, 5mM MgCl ₂ ) containing reagent 50μ
And digest with EcoRI (NEBiolabs, 4 units) for 1 hour at 37 ° C. A rennin fragment (about 350 bp) is isolated from a 2% agarose gel. Charge 293-118 / 37 RFI D
NA (5μ) with Hind III (NEBiolabs, 4 units) 37
Cleave for 1 hour at 50 ° C. in 50 μl of Reagent containing Buffer H. After performing phenol extraction and ethanol precipitation, the DNA was treated with T ₄ DNA polymerase (P-
Buffer T50μ using L Biochemicals, 10 units)
Make blunt ends in. Next, the DNA was extracted once again with ethanol and precipitated with ethanol, and then the DNA was digested with E.
Buffe with coRI (NEBiolabs, 4 units) for 1 hour at 37 ℃
Cleavage in 50μ of reagent containing rR. 293
EcoRI to Hind III (blunt) DNA fragment from −118/37 (approx.
660 bp) is isolated from a 2% agarose gel. Plasmid DNA (5 μg) from plasmid pGL101 was added to Pvu I
I (NEBiolabs, 4 units) for 1 hour at 37 ℃, Buffer P
Cleavage in 50μ, then the DNA is phenol extracted and ethanol precipitated. Using bovine intestinal alkaline phosphatase (0.1 unit, Boehringer / Mannheim)
After treatment in 50 μC of Buffer C, the DNA is again phenol extracted and ethanol precipitated. 14 ° C for binding reaction
18 hours at linkered-rennin DNA (nucleotides 379-731) 0.2 pmol, Phage 293-118 / 37
Using 0.2 pmoles of rennin DNA (nucleotides 732 to 1456) from p.
Buffer 20 using 300 units of ₄ DNA ligase (NEBiolabs)
performed in μ. Reactant 5μ is used to transform 100μ of CGE4 cells treated with Ca ⁺⁺ (see above). Restriction endonuclease analysis of plasmid DNA from several ampicillin resistant colonies picked from tryptone plates containing 20 μg / ml ampicillin showed that plasmid pCGE18 carrying the rennin sequence appropriately positioned to express the lactose operon promoter.
One colony containing 8 is shown.

We have a lysate of cells carrying the plasmid
By subjecting to a radioimmunoassay using iodinated authentic purified rennin and anti-rennin serum,
Determine the amount of rennin synthesized. The size of the rennin product is determined by electrophoresing immunoprecipitates of ³⁵ S-methionine labeled cell extracts on SDS-containing polyacrylamide gels.

In addition, the amount of active rennin present in E. coli cells is
Standard milk coagulation analysis (B.Foltmann, Methods in Enzymology
Vol. 19, pp. 421-436 [1970]).

8b. Expression of methionine-rennin A in E. coli Rennin A immediately preceded by the initiation codon ATG and under the control of the lactose operon promoter.
A plasmid containing the nucleotide sequence of the gene is made as follows. This construction requires the creation of three separate plusimides that are used in sequential recombination to generate the final product.

The first plasmid to be constructed contains the initiation codon ATG immediately adjacent to the first about 350 nucleotides of the rennin gene. Double-stranded recombinant fl fusion 2
93-118 / 37 DNA (200 μg) restriction endonuclease Ps
Buffe with t I (NEBiolabs, 20 units) at 37 ° C for 150 minutes
Digested in 100μ of reagent containing rP. Tris ° HC
l (pH7.5) 11μ and EcoRI (Boehringer / Mannheim,
80 units / μ) was added and the digestion was continued for another 60 minutes at 37 ° C. The restriction reaction is terminated by adding 1/10 volume of 200 mM EDTA and the DNA restriction fragment is subjected to agarose gel electrophoresis in a 0.6% agarose gel containing 40 mM Tris acetate (pH 8.3). Separated by.
Stain the gel with ethidium bromide (0.5 μg / ml) and
The part containing the 00 bp band was visualized under long-wavelength ultraviolet light and cut out. DNA was separated from the gel by freeze-thaw method and ethanol precipitated. Redissolve DNA in water, restriction endonuclease MspI (NEBiolabs, 30 units)
At 37 ° C for 1 hour, 10 mM Tris ° HCl (pH7.4), 10 mM Mg
Digested in 50μ of reagent containing Cl ₂ , 6 mM KCl and 1 mM dithiothreitol. This reaction produced a fragment containing the sequence 5'CGCG TTC ▲ ▼ GAG …… CG AAG CCC CTC …… 5 'near the start of the rennin gene (mark the first codon of the rennin gene sequence. However, this is the nucleotide in Table 1.
379 to 381 are shown). After performing phenol extraction, ether extraction and ethanol precipitation, this fragment and the two smaller fragments generated by MspI digestion were used for E. coli DNA polymerase I (Boehringer / Mannheim, 3 units).
Klenow fragment of 0.05 mM Deoxyadenosine Triphosphate, 6.6 mM Tris ° HCl (pH 7.5) 6.
Treated in 42μ of reagent containing 6 mM NaCl, 6.6 mM MgCl ₂ and 6.6 mM dithiothreitol. This reaction shaped the two nucleotides from the above sequence to generate 5'CGGC TTC GAG ... AAG CCC CTC ... 5 '. After performing phenol extraction and ether extraction, deoxyadenosine triphosphonate was added,
Separated from high molecular weight species by adding 0.5 mM 200 mM spermine, incubating on ice for 15 minutes and centrifuging for 10 minutes in a microcentrifuge at 4 ° C to produce 75% pellets. Centrifuge twice in the presence of ethanol at 4 ° C. (5 minutes each). Spermine was removed by addition of 1 ml 75% ethanol, 0.3 M sodium acetate and 10 mM magnesium acetate, incubated for 1 hour on ice and centrifuged as above.

A second treatment of DNA with the Klenow fragment of E. coli DNA polymerase was performed as described above, except that 0.05 mM deoxycytidine triphosphonate was used instead of 0.05 mM deoxyadenosine triphosphonate in the previous reaction. It was By this method, a fragment having the sequence 5'C GGC TTC GAG ... CCC CTC ... 5'in the vicinity of the start point of the rennin gene was produced. After phenol extraction, ether precipitation, and ethanol precipitation, the DNA was redissolved in water and sl nuclease (Boehninger / Mannheim, 100
Reagent 50 containing Buffer S at room temperature for 30 minutes
Treated in μ. This enzyme removed the 5'single-stranded DNA from the fragment to leave the sequence 5'GAG ... CCC CTC ... 5 '.
Therefore, the start of the rennin sequence is at the 5'end of the DNA fragment. Cla I restriction site, and nucleotide A
Synthetic oligonucleotide containing an end having TG (ie, CATCGATG, Collaborative Research, 5μ
g) was kinased with α ³² P-ATP using T ₄ polynucleotide kinase (PL Biochemicals, 3 units) at 37 ° C. for 30 minutes in 25 μl of a reagent containing Buffer Y. The kinased linker (about 200p mol), T ₄ DNA
Buffel overnight at 15 ° C with ligase (NEBiolabs, 900 units)
Treated by incubating in L
It was attached to a DNA fragment. The reaction was terminated by heating at 65 ° C for 5 minutes. 10 x Cla I buffer (1 x = 10 mM
Tris ° HCl (pH8) 10 mM MgCl ₂ ) 4μ, and restriction endonuclease Cla I (Boehringer / Mannheim, 27 units)
Was added. The resulting mixture was incubated at 37 ° C for 1 hour. 4 μ was removed for analysis on a polyacrylamide gel and 10 μ Cla I buffer 1 μ, Cla I enzyme 10 μ, and water 3 μ were added. This mixture was further incubated for 1 hour. Treated DNA containing the desired rennin sequence was purified by separation in a 2% agarose gel containing 40 mM Tris ° acetate buffer (pH 8.3). DNA was visualized by long wavelength UV and removed from the gel by the freeze-thaw method described above. This fragment was then subjected to insertion into a suitable vector.

Reactant containing Cla I buffer was used to generate vector DNA.
Cla I endonuclease (Boehr
inger / Mannheim) was initiated by digesting 3.3 μg of pBR322 DNA with 5.4 units. After performing phenol extraction, ether extraction, and ethanol precipitation, vector DN
Calf intestinal phosphatase (Boehringer / Mannhei
m) 0.06 units treated in Buffer C for 15 minutes at 37 ° C. After phenol extraction, ether extraction, and ethanol precipitation, approximately 1 pmol of vector DNA was mixed with 2 pmol of rennin fragment DNA as described above. These two DNAs
Fragments were bound together in reactant 29μ and T ₄ DNA ligase containing Buffer L (NEBiolabs, 450 units) and. E. coli CGE43 strain (F-V) (lac-pro) XIII, L
Cells having a conversion ability (known as G90 strain) were prepared by the method described in the above item 4 and converted with the ligated DNA. Ampicillin resistant colonies selected on the plates were picked and the plasmid DNA was analyzed by digestion with restriction enzymes. It is necessary to ensure that the sequence part of these plasmids gives the correct structure containing the linker sequence, CATCGATG, flanking the start of the rennin gene sequence GAG. pC
A plasmid with the desired correct sequence, called GE301, was then used in combination with the other two plasmids described below to create the final plasmid that directs expression of methionine-rennin in E. coli. To do.

To generate the second of the three plasmids required for this structure, recombinant phage 293-118 / 37 was used.
Subcloning of the rennin-containing Hind III fragment of double-stranded DNA is required. Double-stranded fl Phage 293-118 / 37 DNA3
µg of restriction endonuclease Hind III (NEBiolab
s, 3 units) for 1 hour at 37 ° C. in 10 μl of a reagent containing Buffer H and 7 mM 2-mercaptoethanol. Add 4 μ of Hinc II (NEBiolabs, 3 units),
The mixture was incubated at 37 ° C for an additional hour.
After phenol extraction, ether extraction, and ethanol precipitation, about 1.5 μg of this DNA was added to pBR322 DNA (Hind II
I and pre-treated with bovine intestinal alkaline phosphatase) and about 1 μg and mixed. 2 DNs
The A fragments were ligated together at 16 ° C. overnight in 20 μl of a reagent containing Buffer L and T ₄ DNA ligase (NEBiolabs, 600 units). 5 μ of this mixture was used to transform cells of the E. coli CGE6 strain and ampicillin resistant colonies were then isolated as described above. Cleavage with restriction enzymes and agarose gel electrophoresis revealed that the resulting plasmid pCGE302 consisted of the prorennin gene sequence from Phage 293-118 / 37 inserted at the Hind III site of pBR322.

The third type required for the construction of the rennin-producing plasmid.
Ingredients, pGL101 (L. Guarente, G. Lauer, TM Roberts and
M. Ptashne [1980], supra) DNA is digested with the restriction endonuclease Pvu II (NEBiolabs, 5 units) for 1 hour at 37 ° C. in 20 μl of a reagent containing Buffer H and 10 mM 2-mercaptoethanol. Needed. After performing phenol extraction, ether extraction, and ethanol precipitation, the DNA was mixed with a kinased synthetic oligonucleotide (CATCGATC, Collaborative Research, about 200 pmol), and T ₄ DNA ligase (NEBiolabs, 900 units) was used. Reactant 30μ containing Buffel L at 16 ° C overnight
Bound in. The reaction was terminated by treating at 65 ° C for 5 minutes. 5 μ of this mixture was used to transform CaCl ₂ treated cells of E. coli CGE43 strain. Plasmid DNA was prepared from several transformants and then digested with restriction enzymes and agarose to identify the desired plasmid pCGE303, which is identical to plasmid pGL101 except that the Pvu II site is converted to the Cla I site. -Subjected to gel electrophoresis.

Construction of the final plasmid containing the ATG-rennin sequence under translational control of the lactose operon promoter involved in vitro recombination of the three plasmids described above, theoretically summarized as follows. To be done. Restriction of plasmid pCGE301 with restriction endonucleases Kpl I and Hind I
It is digested with II and the resulting fragment is treated with calf intestinal alkaline phosphatase. Plasmid pCGE302 is digested with the restriction endonucleases KpnI, HindIII and BglII. The fragments resulting from these two treatments are then mixed and ligated. This DNA is used to transform E. coli CGE43. The major ampicillin resistance plasmid product is pCGE304 which contains ATG linked to the entire rennin coding sequence.

Then the plasmid pCGE303 was added to the restriction endonuclease P
Digested with st I and Cla I and treated with calf intestinal alkaline phosphatase. The plasmid pCGE304 is also P
Digest with st I and Cla I. The DNA fragments generated from these two treatments are mixed together, ligated and used to transform the CGE43 strain. Ampicillin resistance plasmids obtained from transformed cells are analyzed by size, restriction enzyme digestion and DNA sequence to find the desired plasmid pCGE305, which has the ATG rennin sequence under translational control of the lactose operon promoter. This plasmid, when present in E. coli, directs the synthesis of methionine-rennin.

9. Method for obtaining expression of pre-prorennin, prorennin, and rennin in S. cerevisiae These three types, pre-prorennin, methionine-
Prorennin and methionine-valine-rennin are
It is expressed in S. cerevisiae using the promoter and other transcriptional and translational control regions from the S. cerevisiae uracil 3 gene. The yeast uracil 3 gene was partially deleted from the β-galactosidase or lacZ gene. The plasmid (selected,
And on a shuttle vector that can be maintained in yeast or E. coli. This is M.Ros
Proc.Na by e, MJ Casadaban, and DB Botstein.
Class III deletion part # 35 reported in t.Acad.Sci.USA, 78, 2460-2464 (1981). For this plasmid, expression of β-galactosidase activity in yeast is under the control of the uracil 3 gene control region. we,
Pre-prorennin and methionine in S. cerevisiae
This deletion # 35 is used as follows to obtain prorennin, and methionine-valine-rennin expression.

First, a more complete deletion of the uracil 3 coding sequence is obtained by cutting the closed DNA from deletion # 35 with the restriction endonuclease BamHI which cuts at the ura3-lacZ junction. The DNA is excised with the nuclease Bal31 which removes an average of 200 bp. Next, BamH
An I synthetic oligonucleotide linker (CRI) was attached to its ends and the DNAs were attached to each other so that a population of plasmids is present with BamHI sites at various distances from the uracil control region. Gel electrophoresis of purified plasmid DNA cut with restriction enzymes shows plasmid pCGS210 containing a very small ura3 coding sequence. Max
Sequencing from the BamHI site by the method of am and Gilbert proves this. So suitable, Bal
Purify the DNA from the cloned plasmid excised in. This DNA is a marker (marker) capable of selecting Escherichia coli (ampicillin resistance) and yeast [Leu2 basic nutrition (Prototrophy)], an origin of replication of E. coli and yeast,
All of these are plasmid pRB45 (M.Rose, MJCasada
ban, D. Botstein, supra), and the ura3 regulatory region with less than 50 nucleotides of ura3 coding material. In particular, one of these plasmids, called pCGS210, has only 10-20 nucleotides of ura3 encoding material, as determined by DNA sequencing by the method of Maxam and Glbert.

DNA coding for the 5'ends of pre-prorennin, prorennin and rennin can be obtained as follows. The DNA coding for the ATG translation initiation codon and the pre-prorennin gene containing less than 20 nucleotides to the 5'of ATG is fl Charge Bank (ban
k), screening those phages by using restriction enzymes in combination with gel electrophoresis and the Sanger method (F.
Sanger et al. J. Mol. Biol. 143, 161-178 [1981]). The DNA encoding for prorennin with the ATG translation initiation codon is obtained from plasmid pCGE181 described in item 7 above. ATG codon and GTG valine codon for translation initiation (charge 29
The DNA encoding for rennin having 3-207-101) is described in the previous item 8a, rennin DNA-fl charge bank.
Obtained from or for methionine-rennin
The encoding is obtained from the plasmid pCGE304 described in 8b above.

To obtain expression of each of these DNA fragments in yeast,
The following experiment is conducted. The DNA coding for pre-prorennin, methionine-prorennin, methionine-valine rennin or methionine-rennin is cleaved from among the appropriate phage or plasmids described above. The fragment is further digested with Sma I and the desired coding fragment for the rennin form is purified by gel electrophoresis. Similarly, D for the'ZYA fragment from E. coli
BamHI (blunt) -SalI fragment of NA coding (gene for lactose permease (perm and βm-galactosidase excluding 22bp from its 5'end
ease) and lactose transacetylase) pRB45 (M.Rose, MJCasadaban, and D.Bots
tein [1981], see above). Then, the above plasmid pCGS210 was cleaved at the unique BamHI site and excised at a short distance with Bal31 nuclease (i.e., L. Guarente, G. Lauer, M. Ptashen [1980] see above, conditions). Used), thereby all residual ura3
Just remove the coding sequence but not the control sequence
Generates a fragment that has lost DNA. This DNA is also cut with Sal I and then the largest fragment is gel purified. The three-molecule ligation reaction was digested with this vector fragment plus the Sal I-BamH I (blunt) fragment from pRB45 plus Sma I and gel-purified pre-prorennin, methionine-
It is carried out using either prorennin or methionine-valine-rennin DNA. A portion of this ligation reaction is used to transform E. coli CGE4 strain and red colonies on Macconkey lactose and ampicillin plates are picked. Isolation of plasmid DNA and restriction enzyme analysis confirms the structure of the desired plasmid. Conversion into yeast CGY80 strain (see below), leucine prototrophy (prototro
phy), and uracil (excess or limited amount), leucine and X-gal (5-bromo-4-) in minimal medium.
Chloro-3-indolyl-β-D-galactoside, Bach
blue, screened on medium supplemented with em, Calif.) shows that the plasmid directs the translation of the appropriate rennin-β-galactosidase fusion protein under uracil control. Finally, the β of the desired plasmid
-The galactosidase-encoding part is
Cleave with II and Sal I, then remove the largest fragment by gel purification. This fragment is 293
BglII-HindIII from -118/37 (which has been converted to a SalI site with a synthetic oligonucleotide linker, CRI), was ligated to it, thereby restoring the complete pre-prorennin, prorennin or rennin gene. Reproduce. These plasmids direct the translation of pre-prorennin, methionine-prorennin, methionine-valine-rennin or methionine-rennin in the yeast S. cerevisiae.

10. Expression of the prorennin fusion protein in yeast Only 11 nucleotides of the ura3-encoding material
Above (and M. Rose, MJ Casadaban and D. Botstein, 1981, Pro, except that it remains in front of the BamHI site.
c.Nat.Acad.Sci.USA, Vol. 78, pp. 2460-2464), deletion p.
n, submitted for publication. ), The plasmid pCGS28 containing the gene for prorennin fused to the 11 nucleotides of the ura3-encoding material such that the fusion protein is synthesized in S. cerevisiae. Created. In this fusion protein, the first four amino acids of prorennin are methionine-serine-lysine-
It consists of an authentic prorennin molecule, except that it is replaced by alanine. Activation to produce rennin results in loss of the first 42 amino acids of prorennin, so that these first 4 amino acids have no effect on the final rennin product. Details of the structure of this plasmid are described below.

For efficient expression of prorennin in yeast, ur
The a3 gene promoter region was used. This DNA sequence has been cloned and is valid for plasmids (M. Rose, MJ Casadaban and D. Botstejn, 1981, Proc. N.
at.Acad.Sci.USA 78, 2460-2464.) The plasmid pRB71 obtained from M. Rose has a ura3 promoter region,
And having 11 nucleotides of the uracil 3 gene linked to a fragment of the lacZ gene that omits the first 22 nucleotides. The junction between the two defective genes is
BamHI restriction endonuclease site. This plasmid also contains the yeast plasmid 2 μm, the leu2 gene from yeast, and the origin of replication plus the ampicillin resistance gene from pBR322, as described by M. Rose et al. (See above). Thus, the plasmid can be grown and its presence can be selected for in either E. coli or S. cerevisiae.

Two basic plasmid constructs were generated to obtain expression of the prorennin-binding protein in yeast (ie, fused to the first 11 nucleotides of the ura3 gene and regulated by the ura3 promoter). Let The first structure is a ura3-prorennin-lacZ fusion that produces an active β-galactosidase fusion protein when placed in yeast, indicating that the ura3 promoter directs transcription of the desired fusion gene. There is. The second structure is the replacement of the lacZ portion with the rest of the prorennin, which occurs in yeast cells which produce a prorennin molecule with four amino acids characterized by the ura3 gene.

In the initial structure, the 5'portion of the prorennin gene is inserted in the BamHI site such that ura3, prorennin, and lacZ are all in the same translational reading frame. This is done as follows. Double-stranded recombinant fl phage 293-118 / 37D having the complete prorennin gene
NA (12 μg) was digested with Sma I restriction endonuclease (NEB
iolabs) 7 units, 2 hours at 37 ℃, 20mM KCl 6mM Tris
Digestion was carried out in 50 μl of a reagent containing HCl (pH 8) 6 mM MgCl ₂ and 6 mM 2-mercaptoethanol. The DNA was phenol extracted, ether extracted, and ethanol precipitated. Next, a BamHI synthetic oligonucleotide linker (CRI, C) which had been phosphorylated at its 5'end using the T ₄ polynucleotide kinase described in 7 above.
CGGATCCGG) 250 pmol at 14 ° C overnight in Buffer D and T ₄ D
It was ligated to BamH I-cleaved forage DNA in 40μ of a reagent containing NA ligase (NEBiolabs). Following binding, the reagents were added to 180 mM NaCl, 7 mM MgCl ₂ and 5 mM T.
Dilute with 5 volumes of buffer containing ris ° HCl (pH 8) to 65
The mixture was heated at 0 ° C. for 5 minutes, cooled on ice, and added with 15 units of BamHI endonuclease per hour for digestion for 3 hours. After phenol extraction, ether extraction, and ethanol precipitation, the DNA was redissolved in water and electrophoresed in a 2% agarose gel. The DNA was eluted from the band corresponding to about 440 bp of BamHI-SmaI (BamHI-linkered) fragment by rinsing the frozen gel fragment and collecting the remaining liquid (freeze-thaw method). The DNA fragment was ethanol precipitated and redissolved in 6μ of water. Plasmid pRB7
About 5 μg of 1 DNA was added to BamH I endonuclease (NEBiolab
s) 20 units were digested for 2 hours at 37 ° C. in 50 μl of Reagent containing Buffer X and 6 mM 2-mercaptoethanol. After performing phenol extraction, ether extraction, and ethanol precipitation, the DNA was redissolved in 20 μl of water, and calf intestinal alkaline phosphatase (Boehringer / Mannh
eim) 0.1 unit at 37 ° C. for 30 minutes in 50 μl of Reagent containing Buffer C. After phenol extraction and ethanol precipitation, the DNA was redissolved in 6 μm water and BamHI-S
It was added to the binding reagent containing 6 μl of amI (BamHI-linkered) fragment. The ligation reaction was performed using T ₄ DNA ligase (NEBiola
bs) 600 units, performed in buffer L 20μ overnight at 14 ° C. Cells of Escherichia coli CGE6 strain were transformed with the ligated DNA to obtain ampicillin-resistant transformants as described above. About 200 transformants were subjected to the colony hybridization method of M. Grunstein and DS Hogness (1975, Proc. Nat. Acad.sci. USA Vol. 72, 3961-396.
Yotsute to page 5), was labeled with ^alpha-32 P as a probe (probe). Notch (nick) Translated Recombinant Phage 29
Tested with 3-207 DNA. Almost 20% of the transformants contained a rennin sequence according to this criterion. Plasmid DNA was prepared from 10 transformants and the insert was placed in Pst I
It was determined from the pattern of fragments generated by digestion with endonucleases. One of the plasmids pCGS16, which contained the prorennin fragment in the correct orientation, was cloned into A. Hinne
n, JB Hicks and G. Fink's report (1978, Proc. Nat. Aca
S. cerevisiae CGY80 strain (NATa, leu2-3, leu2-112, his3, trpl-28) according to D. Sci. USA Vol. 75, pp. 1929-1933).
9, ura3-52). Due to the presence of the leu2 gene on the plasmid, a yeast transformant capable of growing without the addition of leucine was used as a chromogenic substrate X-gal.
Streaked onto minimal medium plates containing (as described above by M. Rose et al.) And supplemented with uracil, tryptophan and histidine. All the transformations tested gave blue colonies on X-gal minimal medium, indicating that β-galactosidase was produced. This means that the ura3-prorennin-lacZ fusion protein is produced and that the translational reading frames for each of the three protein fragments are identical.

This result suggested that the same plasmid directs the expression of the ura3-prorennin fusion protein if the β-galactosidase sequence is replaced by the rest of the prorennin gene. Therefore, plasmid pCGS16 8 μg was added to Bg
l II restriction endonuclease (NEBiolabs) at 37 ° C
Digested in Buller P80μ for 1 hour. Then 1M N
10 μl of aCl and 6 μl of water were added to the reactants and the DNA was further digested with 16 units of Sal I endonuclease for 1 hour at 37 ° C. After phenol extraction, ether extraction, and ethanol precipitation, the DNA was treated with 0.06 units of calf intestinal alkaline phosphatase (Boehringer / Mannheim) for 15 minutes at 37 ° C in 50 μl of a reagent containing Buffer C. . The reaction was terminated by phenol extraction of DNA and ethanol precipitation.

On the other hand, recombinant fl Phage 293-118 / 37 double-stranded DNA about 15 μg
12 units of Hind III (NEBiolabs) at 37 ° C for 2 hours, B
Cut in uffer H 100 μ. After phenol and ether extraction and ethanol precipitation, DNA
(6 μg) of E. coli DNA polymerase (Boehringer / Mann
Heim) 10 units at 10 ° C. for 10 minutes in Buffer D 40 μ to make smooth ends. Next, the Sal I synthetic oligonucleotide linker (CRI, GGT) that had been phosphorylated using T ₄ polynucleotide kinase as described above was used.
CGACC) 250 pmol was added along with sufficient buffer components to keep the concentrations of all components constant. Linker, T ₄
The DNA was ligated onto the DNA by incubating overnight at 900C with 900 units of DNA ligase (NEBiolabs). Next, 10 mM Tris HCl (pH 8), 10 mM MgCl ₂ and
5 volumes of buffer consisting of 180 mM NaCl were added and the solution was adjusted to 65
Heat at 5 ° C for 5 minutes, cool on ice, then add Sal I restriction endonuclease (NEBiolabs) at 20 units per hour and incubate at 37 ° C for 5 hours.
The DNA was extracted with phenol, extracted with ether, precipitated with ethanol, and then redissolved in 20 μl of water.
(NEBiolabs) 5 units were digested for 1 hour at 37 ° C. in 30 μl of Reagent containing Buffer P. Then react 20
Finished with 1/10 volume of 0 mM EDTA and run on a 2% agarose gel. Bal II-Hind III (Sal I-linkered) fragment About 10
The band corresponding to 00 bp was cut out and the DNA was recovered by the freeze-thaw method described above.

Ethanol-precipitated pCGS ₁₆ DNA, which had been digested with Bgl II and Sal I endonucleases, was gel purified 2
The 93-118 / 37Bgl II-Hind III (SalI-linkered) DNA fragment was redissolved in 13 μl of water and the two fragments
The fferL and T ₄ DNA ligases (NEBiolabs) were ligated together in 20 μl of a reaction containing 600 units. Cells of strain CGE ⁶ were treated with CaCl ₂ and converted with ligated DNA as described above. Plasmid DNA was purified from 5 different ampicillin resistant transformants and subjected to digestion with BamHI or PstI plus SalI. The band part in 2% agarose gel is
The complete prorennin sequence was shown to be present in plasmid pCGS28.

Therefore, the yeast CGY 80 strain was added to A. Hinnen, JBHicks, and GF.
Leucine prototrophs were selected by conversion with plasmid DNA by ink (1979, see above).
One such transformant CGY116, ³⁵ S for 1/2 generation
By growing in a typical phase at 30 ° C. in a minimal medium containing an appropriate amino acid supplement labeled with 100 μci of L-methionine, and vortexing them with glass beads (250-300 μm) for 3 minutes. Dissolved. The extracts were clarified by centrifugation and immunoprecipitated with rennin antiserum. The immunoprecipitate was dissolved in SDS sample buffer and contained 0.1% SDS 1
UK Laemmli and M. in a 0% polyacrylamide gel.
It was electrospun according to Farre's method (see above).
Autoradiography has plasmid pCGS28
It was shown that the CGY116 strain directs the synthesis of a protein that reacts with the rennin antiserum and is of the expected size for prorennin. Furthermore, the presence of excess unlabeled rennin during the immunoprecipitation procedure abolishes the radioactive band of prorennin. Therefore, S cerevisiae
The CGY116 strain produces calf prorennin fused to 4 amino acids from the yeast ura3 gene instead of the first 4 amino acids of prorennin. The standard method described by B. Foltman (Methods in Enzymology Vol. 19, 42
Activation of the ura3-prorennin fusion protein by pp. 1-436, 1970) occurs during activation of the "pro" zymogen (enzymatic) peptide (which in this case contains four "foreign" amino acids). Being cleaved produces active rennin which is identical to authentic calf renni A. Plasmid pCGS28
CGY116 stocks with American Type Culture
It has been deposited with the Collection (ATCC) and its accession number is 20623.

CGY116 strain is S cerevisiae CGY80 strain (MATa.leu2-3, le
u2-112, ura3-52, his3V, trp1-289, plasmid pCGS28
It has). Plasmid pCGS28 is defined as follows (see Figure 6). That is, most of the plasmids are pBR322 (JGSutcliffe [1979] Cold Spring H).
arbor Symposium Vol. 43, pp. 77-90), EccRI, yeast 2μ
m plasmid (JL Hartley and JEDonelson [198
0], Nature 286, 860-865), LEU2 gene (A. Hinnen, J. Hicks and GR Fink [1978] Proc. Nat. A.
caD.Sci.USA, Vol. 75, pp. 1929-1933), SalI-XhoI fragment of yeast chromosomal DNA, ura3 gene (11 nucleotide 3'site of translation initiation from BamHI as in pRB71), plus nucleotide It contains prorennin A from the BamHI site at # 267 to the end of the gene.

Referring again to Table 1, the nucleotide and amino acid sequences for preyprorennin A are illustrative of nucleotide sequences for some materials of the invention. These materials can be cleaved from the presented nucleotide sequences by conventional methods. Similarly, preyprorennin form A has an aspartate residue at position number 290 (corresponding to 1108 to 110 of the nucleotide sequence in Table 1 and corresponding to the 286th position from Ala in the prolene amino acid sequence) by Foltmann et al. Can be converted to prorennin form B by using glycine at this position instead of. Table 1
A useful product obtained from preyprorennin derived by the method of the present invention, shown in, contains an amino acid sequence corresponding to the following nucleotide sequence forming part of the recombinant DNA material.

1. Gene coding for polypeptides exhibiting pre-prorennin activity having the nucleotide sequences numbered 379 to 1350 in Table 1 and as repeated below.

2. Gene coding for polypeptides exhibiting rennin activity having the nucleotide sequences shown in Table 1 as numbers 205-1350 and as repeated below.

3. Gene coding for polypeptides exhibiting rennin activity having the nucleotide sequences numbered 253-1350 in Table 1 and as repeated below.

4. The start of nucleotides 205-252 of Table 1 (initia
tor) Coding of the preyprorennin coding sequence for the 16 amino acids containing the ATG codon. This sequence directs the secretion of pre-prorennin from gastric cells that synthesize pre-pro-rennin, thus this sequence of their protein product from the host cell into the periplasmic space or medium. It may be useful when linked to other cloned genes to direct secretion. In addition, unnecessary for rennin activity,
These extra nucleotides, when translated into amino acids, play a role in stabilizing enzymes that resist proteolysis while in the cell. It can have general utility for the stabilization of cloned gene products in a variety of host cells and shelf items.

5. The "pro" or zymogen sequence at nucleotide numbers 253-378 in Table 1, which is a 126 nucleotide coding sequence for the 42 amino acids that form the zymogen of the prorennin molecule. This sequence forms the inactive zymogen of rennin and is removed to generate active rennin. Inactive zymogens can have a long shelf life. It can also have general utility for stabilizing the rennin molecule and thus the gene product of other cloned genes.

As used herein, the term "genetic material derived from recombinant DNA material" refers to a host cell that has been transformed with recombinant DNA and cloned to obtain cells carrying the genetic information for the desired product. Means the genetic material of. Recombinant DNA material, in its ordinary sense, is used to represent DNA obtained from two or more different sources that are joined or joined together to form a single molecule, It also includes synthetic DNA obtained by, for example, chemical synthesis. Clearly, the recombination method used to isolate and obtain the novel recombinant DNA material is then cloned and produces host cells that can be grown without the need to reuse the genetic recombination method. can do. In such cases, the cloned cells will be obtained from cells initially treated by the recombinant DNA method and will contain genetic material derived from the recombinant DNA material.

As mentioned above, the recombinant DNA molecule is formed to contain gene coding for at least one polypeptide exhibiting milk coagulation activity, or is useful for making such a polypeptide. is there.

Although specific prorennin, pre-prorennin and rennin genes are detailed in Table 1, the terms used herein refer to nucleotide or amino acid sequences, or milk coagulation or catalytic activity of the final rennin product. Those functional equivalen with all changes in the modified sequence that do not have a significant effect.
It should be understood to include ts). Therefore, the broad term "rennin" as used in rennin, pre-prorennin and prorennin is meant to include all sequences of amino acids that coagulate mammalian milk, such as cow or goat milk, Thus, selected fragments of rennin as previously arrayed in the prior art can be included. Rennin, pre-prorennin and prorennin can have non-functional amino acid sequences which can be removed by conventional methods to enhance the desired activity of the polypeptide.

As noted above, calf rennin is present in two allelic forms A and B that differ in the position number according to Foltmann et al. At sequence position 290 and, at best, at position 204. Although the cloning and expression of rennin A is described here, the gene for rennin B can be easily generated from type A by the simple technique summarized below. Expression of rennin B can be obtained in the same manner as described here for type A. To generate the gene for rennin B, oligonucleotides can be chemically synthesized that span the region that is to be altered and contains the desired alteration. For example, 2
Two 20-mer oligonucleotides (one of the sequences identical to nucleotides 847-866 (Table 1) except nucleotide 856 was changed from A to G and nucleotides 1099-1118 (Table 1) except nucleotide 1109). One of the identical sequences was changed from A to G.) and was optionally scored, and RB Wallace et al. (Science [1980] 20th.
9, 1396-1400).
Fl-fusion 293-118 / 37, which had been converted to single-stranded with III
Used to facilitate second DNA strand synthesis of double-stranded DNA. Double-stranded circular DNA produced are combined in T ₄ DNA polymerase, it is used to transform a suitable E. coli strain. The mixture of phages results in several phages carrying the gene for rennin A and some having a modified rennin A gene with one or the other of two characterized changes. The DNA sequence of the appropriate restriction fragment allows the selection of the phage responsible for each desired change, and the rennin B gene can be generated by joining the two genes together at the appropriate restriction sites. it can. If rennin A to be converted to rennin B differs from rennin B only at position 290, it is necessary to use only synthetic oligonucleotides covering the region 1099-1118, 847-8
The array for 66 is not used.

Although the method of the invention describes starting with RNA material, it can also be started by isolating the gene obtained from genomic DNA. In that case, a suitable host organism is used in which the intervening sequences are first joined, or the RNA can be processed to remove the intervening sequences. Chemically synthesizing the appropriate RNA or DNA, or portions thereof, and then using the techniques described herein to ultimately obtain expression of either rennin, preprorennin and / or prorennin You can also leave by train. In addition, a cloned gene for a protein that coagulates milk in other organisms such as sheep, goats, pigs, or buffalo, which produces a rennin-like enzyme, can be prepared using the techniques described herein. Can be generated.

[Brief description of drawings]

Figure 1 shows the plasmid pCGE5, Figure 2 shows the plasmid pCGE5 cut with Hind IV and digested with exonuclease Bal31, Figure 3 shows the plasmid pCGE17, Figure 4 shows the plasmid pCGE21, FIG. 5 shows the arrangement of nucleotides from pBR322 and pGL101 in plasmid pCGE21 and FIG. 6 shows plasmid pCGS28.

Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication (C12N 1/19 C12R 1: 865) (C12N 1/21 C12R 1:19) (C12N 9/64 C12R 1: 19) (C12N 9/64 C12R 1: 865) (72) Inventor Donald Taylor Moir-USA Masachiyu Settsu 02154 Waltham Virginia Road 39 (72) Inventor Allison Taunton Rigby United States Masachiyu Setu 01773 Lincoln Hualer Road (No address) (72) Inventor Gerardo Francis Vouvis United States Masachiyu Setu 02154 Worsum Bacon Street 350 (56) References Special Features Kai 58-9687 (JP, A) JP 58-109499 (JP, A) Proc. Natl. Acad. Sc i. USA. , 1977 [74] P. 2321-2324 Agric. Biol. Chem. , 1980 [44] P. 1373-1381

Claims (3)

[Claims] 1. The following prorennin nucleotide sequence, namely GCT GAG ATC ACC AGG ATC CCT CTG TAC AAA GGC AAG TC.
T CTG AGG AAG GCG CTG AAG GAG CAT GGG CTT CTG GAG
GAC TTC CTG CAG AAA CAG CAG TAT GGC ATC AGC AGC AA
G TAC TCC GGC TTC GGG GAG GTG GCC AGC GTG CCC CTG ACC AAC TAC CTG GA
T AGT CAG TAC TTT GGG AAG ATC TAC CTC GGG ACC CCG
CCC CAG CAG TTC ACC GTG CTG TTT GAC ACT GGC TCC TC
T GAC TTC TGG GTA CCC TCT ATC TAC TGC AAG AGC AAT
GCC TGC AAA AAC CAC CAG CGC TTC GAC CCG AGA AAG TC
G TCC ACC TTC CAG AAC CTG GGC AAG CCC CTG TCT ATC
CAC TAC GGG ACA GGC AGC ATG CAG GGC ATC CTG GGC TA
T GAC ACC GTC ACT GTC TCC AAC ATT GTG GAC ATC CAG
CAG ACA GTA GGC CTG AGC ACC CAG GAG CCC GGG GAC GT
C TTC ACC TAT GCC GAA TTC GAC GGG ATC CTG GGG ATG
GCC TAC CCC TCG CTC GCC TCA GAG TAC TCG ATA CCC GT
G TTT GAC AAC ATG ATG AAC AGG CAC CTG GTG GCC CAA
GAC CTG TTC TCG GTT TAC ATG GAC AGG AAT GGC CAG GA
G AGC ATG CTC ACG CTG GGG GCC ATC GAC CCG TCC TAC
TAC ACA GGG TCC CTG CAC TGG GTG CCC GTG ACA GTG CA
G CAG TAC TGG CAG TTC ACT GTG GAC AGT GTC ACC ATC
AGC GGT GTG GTT GTG GCC TGT GAG GGT GGC TGT CAG GC
C ATC CTG GAC ACG GGC ACC TCC AAG CTG GTC GGG CCC
AGC AGC GAC ATC CTC AAC ATC CAG CAG GCC ATT GGA GC
C ACA CAG AAC CAG TAC GAT GAG TTT GAC ATC GAC TGC
GAC AAC CTG AGC TAC ATG CCC ACT GTG GTC TTT GAG AT
C AAT GGC AAA ATG TAC CCA CTG ACC CCC TCC GCC TAT
ACC AGC CAG GAC CAG GGC TTC TGT ACC AGT GGC TTC CA
G AGT GAA AAT CAT TCC CAG AAA TGG ATC CTG GGG GAT
GTT TTC ATC CGA GAG TAT TAC AGC GTC TTT GAC AGG GC
C AAC AAC CTC GTG GGG CTG GCC AAA GCC ATC TGA, the following pre-prorennin nucleotide sequence, namely ATG AGG TGT CTC GTG GTG CTA CTT GCT GTC TTC GCT CT
C TCC CAG GGC GCT GAG ATC ACC AGG ATC CCT CTG TAC AAA GGC AAG TC
T CTG AGG AAG GCG CTG AAG GAG CAT GGG CTT CTG GAG
GAC TTC CTG CAG AAA CAG CAG TAT GGC ATC AGC AGC AA
G TAC TCC GGC TTC GGG GAG GTG GCC AGC GTG CCC CTG ACC AAC TAC CTG GA
T AGT CAG TAC TTT GGG AAG ATC TAC CTC GGG ACC CCG
CCC CAG GAG TTC ACC GTG CTG TTT GAC ACT GGC TCC TC
T GAC TTC TGG GTA CCC TCT ATC TAC TGC AAG AGC AAT
GCC TGC AAA AAC CAC CAG CGC TTC GAC CCG AGA AAG TC
G ACC TTC CAG AAC CTG GGC AAG CCC CTG TCT ATC CAC
TAC GGG ACA GGC AGC ATG CAG GGC ATC CTG GGC TAT GA
C ACC GTC ACT GTC TCC AAC ATT GTG GAC ATC CAG CAG
ACA GTA GGC CTG AGC ACC CAG GAG CCC GGG GAC GTC TT
C ACC TAT GCC GAA TTC GAC GGG ATC CTG GGG ATG GCC
TAC CCC TCG CTC GCC TCA GAG TAC TCG ATA CCC GTG TT
T GAC AAC ATG ATG AAC AGG CAC CTG GTG GCC CAA GAC
CTG TTC TCG GTT TAC ATG GAC AGG AAT GGC CAG GAG AG
C ATG CTC ACG CTG GGG GCC ATC GAC CCG TCC TAC TAC
ACA GGG TCC CTG CAC TGG GTG CCC GTG ACA GTG CAG CA
G TAC TGG CAG TTC ACT GTG GAC AGT GTC ACC ATC AGC
GGT GTG GTT GTG GCC TGT GAG GGT GGC TGT CAG GCC AT
C CTG GAC ACG GGC ACC TCC AAG CTG GTC GGG CCC AGC
AGC GAC ATC CTC AAC ATC CAG CAG GCC ATT GGA GCC AC
A CAG AAC CAG TAC GAT GAG TTT GAC ATC GAC TGC GAC
AAC CTG AGC TAC ATG CCC ACT GTG GTC TTT GAG ATC AA
T GGC AAA ATG TAC CCA CTG ACC CCC TCC GCC TAT ACC
AGC CAG GAC CAG GGC TTC TGT ACC AGT GGC TTC CAG AG
T GAA AAT CAT TCC CAG AAA TGG ATC CTG GGG GAT GTT
TTC ATC CGA GAG TAT TAC AGC GTC TTT GAC AGG GCC AA
C AAC CTC GTG GGG CTG GCC AAA GCC ATC TGA, and rennin nucleotide sequences, i.e., GGG GAG GTG GCC AGC GTG CCC CTG ACC AAC TAC CTG GA
T AGT CAG TAC TTT GGG AAG ATC TAC CTC GGG ACC CCG
CCC CAG GAG TTC ACC GTG CTG TTT GAC ACT GGC TCC TC
T GAC TTC TGG GTA CCC TCT ATC TAC TGC AAG AGC AAT
GCC TGC AAA AAC CAC CAG CGC TTC GAC CCG AGA AAG TC
G TCC ACC TTC CAG AAC CTG GGC AAG CCC CTG TCT ATC
CAC TAC GGG ACA GGC AGC ATG CAG GGC ATC CTG GGC TA
T GAC ACC GTC ACT GTC TCC AAC ATT GTG GAC ATC CAG
CAG ACA GTA GGC CTG AGC ACC CAG GAG CCC GGG GAC GT
C TTC ACC TAT GCC GAA TTC GAC GGG ATC CTG GGG ATG
GCC TAC CCC TCG CTC GCC TCA GAG TAC TCG ATA CCC GT
G TTT GAC AAC ATG ATG AAC AGG CAC CTG GTG GCC CAA
GAC CTG TTC TCG GTT TAC ATG GAC AGG AAT GGC CAG GA
G AGC ATG CTC ACG CTG GGG GCC ATC GAC CCG TCC TAC
TAC ACA GGG TCC CTG CAC TGG GTG CCC GTG ACA GTG CA
G CAG TAC TGG CAG TTC ACT GTG GAC AGT GTC ACC ATC
AGC GGT GTG GTT GTG GCC TGT GAG GGT GGC TGT CAG GC
C ATC CTG GAC ACG GGC ACC TCC AAG CTG GTC GGG CCC
AGC AGC GAC ATC CTC AAC ATC CAG CAG GCC ATT GGA GC
C ACA CAG AAC CAG TAC GAT GAG TTT GAC ATC GAC TGC
GAC AAC CTG AGC TAC ATG CCC ACT GTG GTC TTT GAG AT
C AAT GGC AAA ATG TAC CCA CTG ACC CCC TCC GCC TAT
ACC AGC CAG GAC CAG GGC TTC TGT ACC AGT GGC TTC CA
G AGT GAA AAT CAT TCC CAG AAA TGG ATC CTG GGG GAT
GTT TTC ATC CGA GAG TAT TAC AGC GTC TTT GAC AGG GC
A DNA characterized by being selected from the group consisting of C AAC AAC CTC GTG GGG CTG GCC AAA GCC ATC TGA. 2. The following prorennin nucleotide sequence, namely GCT GAG ATC ACC AGG ATC CCT CTG TAC AAA GGC AAG TC.
T CTG AGG AAG GCG CTG AAG GAG CAT GGG CTT CTG GAG
GAC TTC CTG CAG AAA CAG CAG TAT GGC ATC AGC AGC AA
G TAC TCC GGC TTC GGG GAG GTG GCC AGC GTG CCC CTG ACC AAC TAC CTG GA
T AGT CAG TAC TTT GGG AAG ATC TAC CTC GGG ACC CCG
CCC CAG GAG TTC ACC GTG CTG TTT GAC ACT GGC TCC TC
T GAC TTC TGG GTA CCC TCT ATC TAC TGC AAG AGC AAT
GCC TGC AAA AAC CAC CAG CGC TTC GAC CCG AGA AAG TC
G TCC ACC TTC CAG AAC CTG GGC AAG CCC CTG TCT ATC
CAC TAC GGG ACA GGC AGC ATG CAG GGC ATC CTG GGC TA
T GAC ACC GTC ACT GTC TCC AAC ATT GTG GAC ATC CAG
CAG ACA GTA GGC CTG AGC ACC CAG GAG CCC GGG GAC GT
C TTC ACC TAT GCC GAA TTC GAC GGG ATC CTG GGG ATG
GCC TAC CCC TCG CTC GCC TCA GAG TAC TCG ATA CCC GT
G TTT GAC AAC ATG ATG AAC AGG CAC CTG GTG GCC CAA
GAC CTG TTC TCG GTT TAC ATG GAC AGG AAT GGC CAG GA
G AGC ATG CTC ACG CTG GGG GCC ATC GAC CCG TCC TAC
TAC ACA GGG TCC CTG CAG TGG GTG CCC GTG ACA GTG CA
G CAG TAC TGG CAG TTC ACT GTG GAC AGT GTC ACC ATC
AGC GGT GTG GTT GTG GCC TGT GAG GGT GGC TGT CAG GC
C ATC CTG GAC ACG GGC ACC TCC AAG CTG GTC GGG CCC
AGC AGC GAC ATC CTC AAC ATC CAG CAG GCC ATT GGA GC
C ACA CAG AAC CAG TAC GAT GAG TTT GAC ATC GAC TGC
GAC AAC CTG AGC TAC ATG CCC ACT GTG GTC TTT GAG AT
C AAT GGC AAA ATG TAC CCA CTG ACC CCC TCC GCC TAT
ACC AGC CAG GAC CAG GGC TTC TGT ACC AGT GGC TTC CA
G AGT GAA AAT CAT TCC CAG AAA TGG ATC CTG GGG GAT
GTT TTC ATC CGA GAG TAT TAC AGC GTC TTT GAC AGG GC
C AAC AAC CTC GTG GGG CTG GCC AAA GCC ATC TGA, the following pre-prorennin nucleotide sequence, namely ATG AGG TGT CTC GTG GTG CTA CTT GCT GTC TTC GCT CT
C TCC CAG GGC GCT GAG ATC ACC AGG ATC CCT CTG TAC AAA GGC AAG TC
T CTG AGG AAG GCG CTG AAG GAG CAT GGG CTT CTG GAG
GAC TTC CTG CAG AAA CAG CAG TAT GGC ATC AGC AGC AA
G TAC TCC GGC TTC GGG GAG GTG GCC AGC GTG CCC CTG ACC AAC TAC CTG GA
T AGT CAG TAC TTT GGG AAG ATC TAC CTC GGG ACC CCG
CCC CAG GAG TTC ACC GTG CTG TTT GAC ACT GGC TCC TC
T GAC TTC TGG GTA CCC TCT ATC TAC TGC AAG AGC AAT
GCC TGC AAA AAC CAC CAG CGC TTC GAC CCG AGA AAG TC
G TCC ACC TTC CAG AAC CTG GGC AAG CCC CTG TCT ATC
CAC TAC GGG ACA GGC AGC ATG CAG GGC ATC CTG GGC TA
T GAC ACC GTC ACT GTC TCC AAC ATT GTG GAC ATC CAG
CAG ACA GTA GGC CTG AGC ACC CAG GAG CCC GGG GAC GT
C TTC ACC TAT GCC GAA TTC GAC GGG ATC CTG GGG ATG
GCC TAC CCC TCG CTC GCC TCA GAG TAC TCG ATA CCC GT
G TTT GAC AAC ATG ATG AAC AGG CAC CTG GTG GCC CAA
GAC CTG TTC TCG GTT TAC ATG GAC AGG AAT GGC CAG GA
G AGC ATG CTC ACG CTG GGG GCC ATC GAC CCG TCC TAC
TAC ACA GGG TCC CTG CAC TGG GTG CCC GTG ACA GTG CA
G CAG TAC TGG CAG TTC ACT GTG GAC AGT GTC ACC ATC
AGC GGT GTG GTT GTG GCC TGT GAG GGT GGC TGT CAG GC
C ATC CTG GAC ACG GGC ACC TCC AAG CTG GTC GGG CCC
AGC AGC GAC ATC CTC AAC ATC CAG CAG GCC ATT GGA GC
C ACA CAG AAC CAG TAC GAT GAG TTT GAC ATC GAC TGC
GAC AAC CTG AGC TAC ATG CCC ACT GTG GTC TTT GAG AT
C AAT GGC AAA ATG TAC CCA CTG ACC CCC TCC GCC TAT
ACC AGC CAG GAC CAG GGC TTC TGT ACC AGT GGC TTC CA
G AGT GAA AAT CAT TCC CAG AAA TGG ATC CTG GGG GAT
GTT TTC ATC CGA GAG TAT TAC AGC GTC TTT GAC AGG GC
C AAC AAC CTC GTG GGG CTG GCC AAA GCC ATC TGA, and rennin nucleotide sequences, i.e., GGG GAG GTG GCC AGC GTG CCC CTG ACC AAC TAC CTG GA
T AGT CAG TAC TTT GGG AAG ATC TAC CTC GGG ACC CCG
CCC CAG GAG TTC ACC GTG CTG TTT GAC ACT GGC TCC TC
T GAC TTC TGG GTA CCC TCT ATC TAC TGC AAG AGC AAT
GCC TGC AAA AAC CAC CAG CGC TTC GAC CCG AGA AAG TC
G TCC ACC TTC CAG AAC CTG GGC AAG CCC CTG TCT ATC
CAC TAC GGG ACA GGC AGC ATG CAG GGC ATC CTG GGC TA
T GAC ACC GTC ACT GTC TCC AAC ATT GTG GAC ATC CAG
CAG ACA GTA GGC CTG AGC ACC CAG GAG CCC GGG GAC GT
C TTC ACC TAT GCC GAA TTC GAC GGG ATC CTG GGG ATG
GCC TAC CCC TCG CTC GCC TCA GAG TAC TCG ATA CCC GT
G TTT GAC AAC ATG ATG AAC AGG CAC CTG GTG GCC CAA
GAC CTG TTC TCG GTT TAC ATG GAC AGG AAT GGC CAG GA
G AGC ATG CTC ACG CTG GGG GCC ATC GAC CCG TCC TAC
TAC ACA GGG TCC CTG CAC TGG GTG CCC GTG ACA GTG CA
G CAG TAC TGG CAG TTC ACT GTG GAC AGT GTC ACC ATC
AGC GGT GTG GTT GTG GCC TGT GAG GGT GGC TGT CAG GC
C ATC CTG GAC ACG GGC ACC TCC AAG CTG GTC GGG CCC
AGC AGC GAC ATC CTC AAC ATC CAG CAG GCC ATT GGA GC
C ACA CAG AAC CAG TAC GAT GAG TTT GAC ATC GAC TGC
GAC AAC CTG AGC TAC ATG CCC ACT GTG GTC TTT GAG AT
C AAT GGC AAA ATG TAC CCA CTG ACC CCC TCC GCC TAT
ACC AGC CAG GAC CAG GGC TTC TGT ACC AGT GGC TTC CA
G AGT GAA AAT CAT TCC CAG AAA TGG ATC CTG GGG GAT
GTT TTC ATC CGA GAG TAT TAC AGC GTC TTT GAC AGG GC
C AAC AAC CTC GTG GGG CTG GCC AAA GCC ATC A bacterium or yeast containing a gene obtained from a DNA selected from the group consisting of and capable of expressing the gene. 3. The following prorennin nucleotide sequence, namely GCT GAG ATC ACC AGG ATC CCT CTG TAC AAA GGC AAG TC.
T CTG AGG AAG GCG CTG AAG GAG CAT GGG CTT CTG GAG
GAC TTC CTG CAG AAA CAG CAG TAT GGC ATC AGC AGC AA
G TAC TCC GGC TTC GGG GAG GTG GCC AGC GTG CCC CTG ACC AAC TAC CTG GA
T AGT CAG TAC TTT GGG AAG ATC TAC CTC GGG ACC CCG
CCC CAG GAG TTC ACC GTG CTG TTT GAC ACT GGC TCC TC
T GAC TTC TGG GTA CCC TCT ATC TAC TGC AAG AGC AAT
GCC TGC AAA AAC CAC CAG CGC TTC GAC CCG AGA AAG TC
G TCC ACC TTC CAG AAC CTG GGC AAG CCC CTG TCT ATC
CAC TAC GGG ACA GGC AGC ATG CAG GGC ATC CTG GGC TA
T GAC ACC GTC ACT GTC TCC AAC ATT GTG GAC ATC CAG
CAG ACA GTA GGC CTG AGC ACC CAG GAG CCC GGG GAC GT
C TTC ACC TAT GCC GAA TTC GAC GGG ATC CTG GGG ATG
GCC TAC CCC TCG CTC GCC TCA GAG TAC TCG ATA CCC GT
G TTT GAC AAC ATG ATG AAC AGG CAC CTG GTG GCC CAA
GAC CTG TTC TCG GTT TAC ATG GAC AGG AAT GGC CAG GA
G AGC ATG CTC ACG CTG GGG GCC ATC GAC CCG TCC TAC
TAC ACA GGG TCC CTG CAC TGG GTG CCC GTG ACA GTG CA
G CAG TAC TGG CAG TTC ACT GTG GAC AGT GTC ACC ATC
AGC GGT GTG GTT GTG GCC TGT GAG GGT GGC TGT CAG GC
C ATC CTG GAC ACG GGC ACC TCC AAG CTG GTC GGG CCC
AGC AGC GAC ATC CTC AAC ATC CAG CAG GCC ATT GGA GC
C ACA CAG AAC CAG TAC GAT GAG TTT GAC ATC GAC TGC
GAC AAC CTG AGC TAC ATG CCC ACT GTG GTC TTT GAG AT
C AAT GGC AAA ATG TAC CCA CTG ACC CCC TCC GCC TAT
ACC AGC CAG GAC CAG GGC TTC TGT ACC AGT GGC TTC CA
G AGT GAA AAT CAT TCC CAG AAA TGG ATC CTG GGG GAT
GTT TTC ATC CGA GAG TAT TAC AGC GTC TTT GAC AGG GC
C AAC, AAC CTC GTG GGG CTG GCC AAA GCC ATC TGA, the following pre-prorennin nucleotide sequence, namely ATG AGG TGT CTC GTG CTA CTT GCT GTC TTC GCT CTC TC
C CAG GGC GCT GAG ATC ACC AGG ATC CCT CTG TAC AAA GGC AAG TC
T CTG AGG AAG GCG CTG AAG GAG CAT GGG CTT CTG GAG
GAC TTC CTG CAG AAA CAG CAG TAT GGC ATC AGC AGC AA
G TAC TCC GGC TTC GGG GAG GTG GCC AGC GTG CCC CTG ACC AAC TAC CTG GA
T AGT CAG TAC TTT GGG AAG ATC TAC CTC GGG ACC CCG
CCC CAG GAG TTC ACC GTG CTG TTT GAC ACT GGC TCC TC
T GAC TTC TGG GTA CCC TCT ATC TAC TGC AAG AGC AAT
GCC TGC AAA AAC CAC CAG CGC TTC GAC CCG AGA AAG TC
G TCC ACC TTC CAG AAC CTG GGC AAG CCC CTG TCT ATC
CAC TAC GGG ACA GGC AGC ATG CAG GGC ATC CTG GGC TA
T GAC ACC GTC ACT GTC TCC AAC ATT GTG GAC ATC CAG
CAG ACA GTA GGC CTG AGC ACC CAG GAG CCC GGG GAC GT
C TTC ACC TAT GCC GAA TTC GAC GGG ATC CTG GGG ATG
GCC TAC CCC TCG CTC GCC TCA GAG TAC TCG ATA CCC GT
G TTT GAC AAC ATG ATG AAC AGG CAC CTG GTG GCC CAA
GAC CTG TTC TCG GTT TAC ATG GAC AGG AAT GGC CAG GA
G AGC ATG CTC ACG CTG GGG GCC ATC GAC CCG TCC TAC
TAC ACA GGG TCC CTG CAC TGG GTG CCC GTG ACA GTG CA
G CAG TAC TGG CAG TTC ACT GTG GAC AGT GTC ACC ATC
AGC GGT GTG GTT GTG GCC TGT GAG GGT GGC TGT CAG GC
C ATC CTG GAC ACG GGC ACC TCC AAG CTG GTC GGG CCC
AGC AGC GAC ATC CTC AAC ATC CAG CAG GCC ATT GGA GC
C ACA CAG AAC CAG TAC GAT GAG TTT GAC ATC GAC TGC
GAC AAC CTG AGC TAC ATG CCC ACT GTG GTC TTT GAG AT
C AAT GGC AAA ATG TAC CCA CTG ACC CCC TCC TAT ACC
AGC CAG GAC CAG GGC TTC TGT ACC AGT GGC TTC CAG AG
T GAA AAT CAT TCC CAG AAA TGG ATC CTG GGG GAT GTT
TTC ATC CGA GAG TAT TAC AGC GTC TTT GAC AGG GCC AA
C AAC CTC GTG GGG CTG GCC AAA GCC ATC TGA, and rennin nucleotide sequences, i.e., GGG GAG GTG GCC AGC GTG CCC CTG ACC AAC TAC CTG GA
T AGT CAG TAC TTT GGG AAG ATC TAC CTC GGG ACC CCG
CCC CAG GAG TTC ACC GTG CTG TTT GAC ACT GGC TCC TC
T GAC TTC TGG GTA CCC TCT ATC TAC TGC AAG AGC AAT
GCC TGC AAA AAC CAC CAG CGC TTC GAC CCG AGA AAG TC
G TCC ACC TTC CAG AAC CTG GGC AAG CCC CTG TCT ATC
CAC TAC GGG ACA GGC AGC ATG CAG GGC ATC CTG GGC TA
T GAC ACC GTC ACT GTC TCC AAC ATT GTG GAC ATC CAG
CAG ACA GTA GGC CTG AGC ACC CAG GAG CCC GGG GAC GT
C TTC ACC TAT GCC GAA TTC GAC GGG ATC CTG GGG ATG
GCC TAC CCC TCG CTC GCC TCA GAG TAC TCG ATA CCC GT
G TTT GAC AAC ATG ATG AAC AGG CAC CTG GTG GCC CAA
GAC CTG TTC TCG GTT TAC ATG GAC AGG AAT GGC CAG GA
G AGC ATG CTC ACG CTG GGG GCC ATC GAC CCG TCC TAC
TAC ACA GGG TCC CTG CAC TGG GTG CCC GTG ACA GTG CA
G CAG TAC TGG CAG TTC ACT GTG GAC AGT GTC ACC ATC
AGC GGT GTG GTT GTG GCC TGT GAG GGT GGC TGT CAG GC
C ATC CTG GAC ACG GGC ACC TCC AAG CTG GTC GGG CCC
AGC AGC GAC ATC CTC AAC ATC CAG CAG GCC ATT GGA GC
C ACA CAG AAC CAG TAC GAT GAG TTT GAC ATC GAC TGC
GAC AAC CTG AGC TAC ATG CCC ACT GTG GTC TTT GAG AT
C AAT GGC AAA ATG TAC CCA CTG ACC CCC TCC GCC TAT
ACC AGC CAG GAC CAG GGC TTC TGT ACC AGT GGC TTC CA
G AGT GAA AAT CAT TCC CAG AAA TGG ATC CTG GGG GAT
GTT TTC ATC CGA GAG TAT TAC AGC GTC TTT GAC AGG GC
C AAC AAC CTC GTG GGG CTG GCC AAA GCC ATC A vector in which a gene consisting of a DNA selected from the group consisting of DNA is effectively linked to a vector that replicates in an appropriate host cell.